Membrane protein thiol cross-linking associated with the permeabilization of the inner mitochondrial membrane by Ca2+ plus prooxidants.
In a previous report (Macedo, D.V., Ferraz, V. L., Pereira-da-Silva, L., and Vercesi, A. E. (1988) in Integration of Mitochondrial Functions (Lemasters, J. J., et al., eds) pp. 535-542, Plenum Publishing Corp., New York), we proposed that the alterations in the inner mitochondrial membrane permeability caused by Ca2+ plus prooxidants could be the consequence of membrane protein sulfhydryl-disulfide transitions. In this study, we show that Ca2+ plus diamide, a thiol oxidant, significantly decrease the ability of beef heart submitochondrial particles to build up and sustain a membrane potential generated by succinate oxidation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of solubilized membrane proteins indicates that these effects on the membrane potential are associated with the production of protein aggregates due to thiol cross-linking. Evidence is also presented that these protein aggregates can be produced in mitoplasts previously loaded with Ca2+ and that this is potentiated by the presence of either diamide or t-butylhydroperoxide. Furthermore, dithiothreitol, a disulfide reductant, was found to be much more effective than NAD(P)+ reductants in reversing Ca2+ efflux induced by prooxidants. It is concluded that the perturbation of the inner mitochondrial membrane caused by Ca2+ plus prooxidants is associated with protein polymerization due to thiol cross-linking, resulting in the production of high molecular mass protein aggregates.
Fagian
M M
MM
Departamento de Bioquimica, Instituto de Biologia, UNICAMP, Campinas S. P., Brazil.
Pereira-da-Silva
L
L
Martins
I S
IS
Vercesi
A E
AE
D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002417:Cattle; D002463:Cell Membrane Permeability / Q000187:drug effects*; D003958:Diamide / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D007425:Intracellular Membranes / Q000502:physiology*; D008564:Membrane Potentials; D008565:Membrane Proteins / Q000378:metabolism*; D008928:Mitochondria / Q000648:ultrastructure*; D008929:Mitochondria, Heart / Q000648:ultrastructure; D008930:Mitochondria, Liver / Q000648:ultrastructure; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D010545:Peroxides / Q000494:pharmacology; D051381:Rats; D013367:Submitochondrial Particles / Q000502:physiology; D013386:Succinates / Q000378:metabolism; D019802:Succinic Acid; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D020122:tert-Butylhydroperoxide
D008565:Membrane Proteins; D010545:Peroxides; D013386:Succinates; D013438:Sulfhydryl Compounds; D003958:Diamide; D004533:Egtazic Acid; D009249:NADP; D020122:tert-Butylhydroperoxide; D019802:Succinic Acid; D002118:Calcium; D004229:Dithiothreitol