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We have investigated pathogenic effects of the tRNA(Lys) 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 V(max) for both the low-affinity and high-affinity phases; however, the K(m) values were not significantly changed. The substrate-dependent synthesis of ATP was decreased to 50% of the control. Analysis of the mitochondrial membrane potential, DeltaPsi, in digitonin-treated cells with tetramethylrhodamine methyl ester (TMRM) with the use of flow cytometry showed a 80% decrease in DeltaPsi at state 4 and an increased sensitivity of DeltaPsi 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 DeltaPsi. Within the range of 87-73% mutated mtDNA, COX activity was decreased to 5-35% and DeltaPsi was decreased to 6-78%. These results demonstrate that the MERRF mutation affects COX activity and DeltaPsi in different proportions with regard to mutation heteroplasmy and indicate that the biochemical manifestation of the MERRF mutation exerts a very steep threshold of DeltaPsi inhibition.

We report a new type of fatal mitochondrial disorder caused by selective deficiency of mitochondrial ATP synthase (ATPase). A hypotrophic newborn from a consanguineous marriage presented severe lactic acidosis, cardiomegaly and hepatomegaly and died from heart failure after 2 days. The activity of oligomycin-sensitive ATPase was only 31-34% of the control, both in muscle and heart, but the activities of cytochrome c oxidase, citrate synthase and pyruvate dehydrogenase were normal. Electrophoretic and western blot analysis revealed selective reduction of ATPase complex but normal levels of the respiratory chain complexes I, III and IV. The same selective deficiency of ATPase was found in cultured skin fibroblasts which showed similar decreases in ATPase content, ATPase hydrolytic activity and level of substrate-dependent ATP synthesis (20-25, 18 and 29-33% of the control, respectively). Pulse-chase labelling of patient fibroblasts revealed low incorporation of [(35)S]methionine into assembled ATPase complexes, but increased incorporation into immunoprecipitated ATPase subunit beta, which had a very short half-life. In contrast, no difference was found in the size and subunit composition of the assembled and newly produced ATPase complex. Transmitochondrial cybrids prepared from enucleated fibroblasts of the patient and rho degrees cells derived from 143B. TK(-)human osteosarcoma cells fully restored the ATPase activity, ATP synthesis and ATPase content, when compared with control cybrids. Likewise, the pattern of [(35)S]methionine labelling of ATPase was found to be normal in patient cybrids. We conclude that the generalized deficiency of mitochondrial ATPase described is of nuclear origin and is caused by altered biosynthesis of the enzyme.

Brown adipose tissue (BAT) is the major thermogenic organ of the human neonate. To determine whether it is also active in the peripheral conversion of T4 to T3, as shown in several animal species, interscapular BAT from 13 newborns of 25-40 weeks gestational age who survived 4 days, at most, was investigated. BAT was found to contain significant amounts of the mitochondrial uncoupling protein (UCP), the rate-limiting component of heat production. The specific content of UCP increased from 29.4 +/- 3.3 to 62.5 +/- 10.2 pmol/mg protein between 25 and 40 weeks of gestation, respectively, and the UCP/F1-ATPase molar ratio, a sensitive marker of brown fat differentiation, increased similarly. BAT was also found to contain iodothyronine 5'-deiodinase (5'D), which appears to be a type II enzyme, based on high affinity for T4 (Km, 2.9 nmol/L) and insensitivity to propylthiouracil (10% inhibition by 1 nmol/L). 5'D was active by 25 weeks gestation, and the specific activity increased from 116 +/- 15 to 417 +/- 46 fmol/h.mg protein during the period examined. The development of 5'D activity was similar to the changes in UCP content; both exhibited a major increase before 32 weeks gestation. The results indicate that thermogenic function and 5'D activity develop in human BAT rather early, during the first half of the last trimester of gestation. The activities of 5'D in human BAT are comparable with 5'D activities found in animal BAT stimulated during the perinatal period, by cold exposure, or by increased cAMP levels.

Iodothyronine 5'-deiodinase (5'D) of mouse brown adipocytes differentiated in cell culture was characterized in detail with respect to the adrenergic control of its biosynthesis. The stimulation of 5'D required mRNA and protein synthesis and was dependent on the stage of differentiation of the cells. The maximum induction was observed around confluence (7-day-old cells), in pre- and post-confluent cells the 5'D activity was significantly less induced. The transient responsiveness of brown fat-cells to the stimulatory effect of adrenergic agents was reflected also in the time course of the induction of 5'D by different concentrations of agonists. The maximum response occurred regularly after an 8 h incubation and implicated a rather fast turnover of the induced enzyme. On the basis of the inhibitory effects of cycloheximide and actinomycin D, the half-life of the induced 5'D and its mRNA were estimated to be 1.5 and 3.3 h respectively. The noradrenaline-induced 5'D activity was shown to be that of the type II enzyme, insensitive to propylthiouracil (PTU). The estimated values of its apparent Km for thyroxine, Km for the co-substrate dithiothreitol, and Vmax. in the presence of 1 mM PTU were 2 nM, 2.6 mM, and 0.1 pmol of I-/h per mg of protein respectively. The 5'D activity was effectively induced by forskolin and dibutyryl cyclic AMP, as well as by isoprenaline, noradrenaline and CGP-12177, but not by phenylephrine, cirazoline or oxymetazoline. This indicates that, contrary to previous observations in vivo, stimulation of 5'D in cultured brown fat-cells involves elevated cyclic AMP levels and is mediated predominantly via beta-receptors, particularly via the so-called beta 3-adrenoceptors.

Different severe disorders of cytochrome c oxidase (COX) have been described in children, but only the defects with autosomal inheritance are suitable for prenatal diagnosis. To perform prenatal diagnosis of fatal infantile COX deficiency a complex approach has been used which combined determination of the genetic origin of the defect, and detailed analysis of the function, content and subunit composition of the enzyme in cultured fetal cells. The tissues and cultured fibroblasts of the patient with Leigh's syndrome showed a COX deficiency of systemic character. The decrease of COX activity to 5–11 per cent was accompanied by proportionally decreased content of the assembled COX enzyme. With the help of transmitochondrial cybrids derived from patient fibroblasts it was proven that the COX defect was of nuclear origin. In a successive pregnancy, the function of oxidative phosphorylation (OXPHOS) was analysed in cultured amniocytes by substrate‐stimulated ATP production and COX activity was compared with the activity of citrate synthase. The amount and composition of OXPHOS complexes was estimated by two‐dimensional (Blue Native/SDS) polyacrylamide gel electrophoresis and was verified immunochemically with specific antibodies. Three independent lines of evidence provided us with reliable data on the function of COX and OXPHOS in fetal cells which were sufficient to rule out the expected enzymatic defect within three weeks after amniocentesis. Copyright © 1999 John Wiley & Sons, Ltd.

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