We have investigated pathogenic effects of the tRNALys A8344G mutation associated with the syndrome myoclonus epilepsy with ragged-red fibres (MERRF) by using fibroblasts and fibroblast-derived cytoplasmic hybrid cells harbouring different percentages of mutated mitochondrial DNA (mtDNA). The activity of cytochrome c oxidase (COX) in patient fibroblasts with 89% mutated mtDNA was decreased to 20% of the control levels. COX exhibited altered kinetics, with a decreased Vmax for both the low-affinity and high-affinity phases; however, the Km values were not significantly changed. The substrate-dependent synthesis of ATP was decreased to 50% of the control. Analysis of the mitochondrial membrane potential, δΨ, in digitonin-treated cells with tetramethylrhodamine methyl ester (TMRM) with the use of flow cytometry showed a 80% decrease in δΨ at state 4 and an increased sensitivity of δΨ 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 between heteroplasmy of the MERRF mutation, COX activity and δΨ. Within the range of 87-73% mutated mtDNA, COX activity was decreased to 5-35% and δΨ was decreased to 6-78%. These results demonstrate that the MERRF mutation affects COX activity and δΨ in different proportions with regard to mutation heteroplasmy and indicate that the biochemical manifestation of the MERRF mutation exerts a very steep threshold of δΨ inhibition.

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