The irreversibility of inner mitochondrial membrane permeabilization by Ca2+ plus prooxidants is determined by the extent of membrane protein thiol cross-linking.
We have previously shown that mitochondrial membrane potential (delta psi) drop promoted by prooxidants and Ca2+ can be reversed but not sustained by ethylene glycol-bis(beta-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) unless dithiothreitol (DTT), a disulfide reductant, is also added [Valle, V. G. R., Fagian, M. M., Parentoni, L. S., Meinicke, A. R., and Vercesi, A. E. (1993). Arch. Biochem. Biophys. 307, 1-7]. In this study we show that catalase or ADP are also able to potentiate this EGTA effect. When EGTA is added long after (12 min) the completion of swelling or delta psi elimination, no membrane resealing occurs unless the EGTA addition was preceded by the inclusion of DTT, ADP, or catalase soon after delta psi was collapsed. Total delta psi recovery by EGTA is obtained only in the presence of ADP. The sensitivity of the ADP effect to carboxyatractyloside strongly supports the involvement of the ADP/ATP carrier in this mechanism. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of solubilized membrane proteins shows that protein aggregation due to thiol cross-linkage formed during delta psi drop continues even after delta psi is already eliminated. Titration with 5,5'-dithio-bis(2-nitrobenzoic acid) supports the data indicating that the formation of protein aggregates is paralleled by a decrease in the content of membrane protein thiols. Since the presence of ADP and EGTA prevents the progress of protein aggregation, we conclude that this process is responsible for both increased permeability to larger molecules and the irreversibility of delta psi drop. The protective effect of catalase suggests that the continuous production of protein thiol cross-linking is mediated by mitochondrial generated reactive oxygen species.
Castilho
R F
RF
Departamento de Bioquímica, Universidade Estadual de Campinas, Brasil.
Kowaltowski
A J
AJ
Vercesi
A E
AE
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D002374:Catalase / Q000378:metabolism; D004229:Dithiothreitol; D004533:Egtazic Acid / Q000494:pharmacology; D008564:Membrane Potentials; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D008930:Mitochondria, Liver / Q000187:drug effects / Q000378:metabolism*; D011487:Protein Conformation; D051381:Rats; D017208:Rats, Wistar; D017382:Reactive Oxygen Species / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000737:chemistry*
D008565:Membrane Proteins; D017382:Reactive Oxygen Species; D013438:Sulfhydryl Compounds; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; D004533:Egtazic Acid; D000244:Adenosine Diphosphate; D002374:Catalase; D002118:Calcium; D004229:Dithiothreitol