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Membrane protein thiol cross-linking associated with the permeabilization of the inner mitochondrial membrane by Ca2+
M M Fagian1, L Pereira-da-Silva, I S Martins
1Departamento de Bioquimica, Instituto de Biologia, UNICAMP, Campinas S. P., Brazil.
Abstract:
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.
Insights
Calcium and oxidants disrupt mitochondrial membrane potential by causing protein aggregation through thiol cross-linking. Dithiothreitol effectively reverses this calcium efflux, indicating a role for disulfide reduction in mitochondrial function.
Area of Science:
- Mitochondrial biochemistry
- Membrane biophysics
Background:
- Previous work suggested Ca2+ plus prooxidants alter inner mitochondrial membrane permeability via sulfhydryl-disulfide transitions.
- Mitochondrial dysfunction is linked to various pathologies.
Purpose of the Study:
- To investigate the role of thiol-disulfide transitions in Ca2+-induced mitochondrial membrane perturbation.
- To identify the molecular mechanisms underlying mitochondrial dysfunction caused by Ca2+ and oxidants.
Main Methods:
- Utilized beef heart submitochondrial particles and mitoplasts.
- Employed Ca2+, diamide (thiol oxidant), t-butylhydroperoxide, and dithiothreitol (disulfide reductant).
- Assessed membrane potential using succinate oxidation and analyzed protein aggregation via SDS-PAGE.
Main Results:
- Ca2+ plus diamide significantly decreased mitochondrial membrane potential.
- SDS-PAGE revealed Ca2+-induced protein aggregates linked to thiol cross-linking.
- Protein aggregation was observed in Ca2+-loaded mitoplasts and potentiated by oxidants.
- Dithiothreitol was more effective than NAD(P)+ reductants in reversing Ca2+ efflux.
Conclusions:
- Ca2+ plus prooxidants perturb the inner mitochondrial membrane by inducing protein polymerization.
- This polymerization results in high molecular mass protein aggregates, disrupting mitochondrial function.
- Thiol cross-linking is a key mechanism in Ca2+-mediated mitochondrial damage.