Defective kinetics of cytochrome c oxidase sold alteration of mitochondrial membrane potential in fibroblasts and cytoplasmic hybrid cells with the mutation for myoclonus epilepsy with ragged-red fibres ('MERRF') at position8344 nt

We have investigated pathogenic effects of the tRNA(Lys) A8344G mutation as sociated with the syndrome myoclonus epilepsy with ragged-red fibres(MERRF) by using fibroblasts and fibroblast-derived cytoplasmic hybrid cells harbo uring different percentages of mutated mitochondrial DNA (mtDNA), The activ ity of cytochrome c oxidase (COX) in patient fibroblasts with 89 % mutated mtDNA was decreased to 20 % of the control levels. COX exhibited altered ki netics, with a decreased V-max for both the low-affinity and high-affinity phases; however, the K-m values were not significantly changed. The substra te-dependent syn thesis of ATP was decreased to 50 % of the control. Analys is of the mitochondrial membrane potential, Delta Psi, in digitonin-treated cells with tetramethylrhodamine methyl ester (TMRM) with the use of flow c ytometry showed a 80 % decrease in Delta Psi at state 4 and an increased se nsitivity of Delta Psi to an uncoupler in fibroblasts from the:patient. The investigation of transmitochondrial cytoplasmic hybrid clones derived from the patient's fibroblasts enabled us to characterize the relationship betw een heteroplasmy of the MERRF mutation, COX activity and Delta Psi. Within the range of 87-73 % mutated mtDNA, COX activity was decreased to 5-35 % an d Delta Psi was decreased to 6-78 %. These results demonstrate that the MER RF mutation affects COX activity and Delta Psi in different proportions wit h regard to mutation heteroplasmy and indicate that the biochemical manifes tation of the MERRF mutation exerts a very steep threshold of Delta Psi inh ibition.