THE IRREVERSIBILITY OF INNER MITOCHONDRIAL-MEMBRANE PERMEABILIZATION BY CA2-PROTEIN THIOL CROSS-LINKING( PLUS PROOXIDANTS IS DETERMINED BY THE EXTENT OF MEMBRANE)

We have previously shown that mitochondrial membrane potential (Delta Psi) drop promoted by prooxidants and Ca2+ can be reversed but not sus tained by ethylene l-bis(beta-aminoethyl-ether)-N,N,N',N'-tetraacetic acid (EGTA) unless dithiothreitol (DTT), a disulfide reductant, is als o 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 t his EGTA effect. When EGTA is added long after (12 min) the completion of swelling or Delta Psi elimination, no membrane resealing occurs un less the EGTA addition was preceded by the inclusion of DTT, ADP, or c atalase soon after Delta Psi was collapsed. Total Delta Psi recovery b y 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-polya crylamide gel electrophoresis of solubilized membrane proteins shows t hat protein aggregation due to thiol cross-linkage formed during Delta Psi drop continues even after Delta Psi is already eliminated. Titrat ion with 5,5'-dithio-bis(2-nitrobenzoic acid) supports the data indica ting that the formation of protein aggregates is paralleled by a decre ase in the content of membrane protein thiols. Since the presence of A DP and EGTA prevents the progress of protein aggregation, we conclude that this process is responsible for both increased permeability to la rger molecules and the irreversibility of Delta Psi drop. The protecti ve effect of catalase suggests that the continuous production of prote in thiol cross-linking is mediated by mitochondrial generated reactive oxygen species.