The A-to-G transition mutation in the tRNALy" gene of mitochondrial DNA (mtDNA), characteristic for the maternally inherited MERRF syndrome (myoclonic epilepsy with ragged red fibers), has been identified by point mutation-specific polymerase chain reaction in extraocular muscle from I1 of 16 healthy people of different ages. No mutation was found in navel-string samples from 5 newborns, in HeLa cells, and in 2 individuals younger than 20 years. On the other hand, the mutation is present in all 5 tested 7C89-year-old individuals and in 6 of 9 2&70-year-old individuals. The amount of mutated from total mtDNA was estimated by 'mispairing PCR' in extraocular muscle of 2 individuals of 74 and 89 years to 2.0 and 2.4%. respectively. In most tissue samples the MERRF mutation occurs together with the 'common deletion' of mtDNA, which was previously shown to accumulate in healthy individuals with increasing age. It is proposed that during aging, deletions and point mutations of mtDNA accumulate, which could impalr mitochondrial energetics.

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The binding of TNP-ATP (2' or 3'-O-(2,4,6-trinitrophenyl)-ATP) to cytochrome c oxidase (COX) from bovine heart and liver and to the two-subunit COX of Paracoccus denitrificans was measured by its change of fluorescence. Three binding sites, two with high (dissociation constant Kd = 0.2 microM) and one with lower affinity (Kd = 0.9 microM), were found at COX from bovine heart and liver, while the Paracoccus enzyme showed only one binding site (Kd = 3.6 microM). The binding of [35S]ATP alpha S was measured by equilibrium dialysis and revealed seven binding sites at the heart enzyme (Kd = 7.5 microM) and six at the liver enzyme (Kd = 12 microM). The Paracoccus enzyme had only one binding site (Kd = 16 microM). The effect of variable intraliposomal ATP/ADP ratios, but at constant total concentration of [ATP + ADP] = 5 mM, on the H+/e- stoichiometry of reconstituted COX from bovine heart and liver were studied. Above 98% ATP the H+/e- stoichiometry of the heart enzyme decreased to about half of the value measured at 100% ATP. In contrast, the H+/e-stoichiometry of the liver enzyme was not influenced by the ATP/ADP ratio. It is suggested that high intramitochondrial ATP/ADP ratios, corresponding to low cellular work load, will decrease the efficiency of energy transduction and result in elevated thermogenesis for the maintenance of body temperature.