Induction of the mitochondrial permeability transition by N-ethylmaleimide depends on secondary oxidation of critical thiol groups. Potentiation by copper-ortho-phenanthroline without dimerization of the adenine nucleotide translocase.
Addition to energized rat liver mitochondria of low micromolar concentrations of the thiol oxidant, copper-o-phenanthroline [Cu(OP)2], causes opening of the permeability transition pore, a cyclosporin A-sensitive channel. The effects of Cu(OP)2 can be reversed by reduction with dithiothreitol (DTT), suggesting that a dithiol-disulfide interconversion is involved. However, at variance with all pore inducers known to act through dithiol oxidation, the effects of Cu(OP)2 are not prevented by treatment of mitochondria with low (10-20 microM) concentrations of N-ethylmaleimide (NEM). Rather, these concentrations of NEM potentiate the inducing effects of Cu(OP)2. We show that this enhancing effect of NEM is blocked by the subsequent addition of DTT, indicating that potentiation by NEM is mediated by an oxidative event rather than by substitution as such. We find that also pore induction by high (0.5-1.0 mM) concentrations of NEM in the absence of oxidants is completely blocked by reduction with DTT or beta-mercaptoethanol. These results underscore the unexpected importance of oxidative events in pore opening by substituting agents. Since we find that pore opening by Cu(OP)2 or by high concentrations of NEM is not accompanied by dimerization of the adenine nucleotide translocase, we conclude that the translocase itself is not the target of the pore-inducing oxidative events triggered by Cu(OP)2 and NEM.
Department of Biomedical Sciences, University of Padua Medical School, Italy.
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Costantini
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Colonna
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Bernardi
D000818:Animals; D002463:Cell Membrane Permeability / Q000187:drug effects / Q000502:physiology*; D016572:Cyclosporine / Q000494:pharmacology; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry; D004229:Dithiothreitol / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology*; D007425:Intracellular Membranes / Q000187:drug effects / Q000502:physiology*; D008623:Mercaptoethanol; D008930:Mitochondria, Liver / Q000737:chemistry*; D000226:Mitochondrial ADP, ATP Translocases; D010084:Oxidation-Reduction; D010539:Permeability / Q000187:drug effects; D010618:Phenanthrolines / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D019163:Reducing Agents / Q000494:pharmacology; D013438:Sulfhydryl Compounds / Q000737:chemistry*; D013439:Sulfhydryl Reagents / Q000494:pharmacology*
D004220:Disulfides; D010618:Phenanthrolines; D019163:Reducing Agents; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; D008623:Mercaptoethanol; D016572:Cyclosporine; D000226:Mitochondrial ADP, ATP Translocases; D005033:Ethylmaleimide; D004229:Dithiothreitol; C025205:1,10-phenanthroline