Two new familial cases of 2-ketoglutarate dehydrogenase (2-KGD) deficiency are reported: a girl who died at 10 years and a boy, still alive at 4 years, born to consanguineous parents. The cases developed progressively severe encephalopathy with axial hypotonia, psychotic behaviour, pyramidal symptoms and failure to thrive. Both children exhibited permanent lactic acidosis with acute episodes during emotional stress and various infections, associated with elevated lactate/pyruvate (L/P) ratio and slightly decreased ketone body ratio in plasma. In fibroblasts, the L/P ratio was greatly increased in the boy. No respiratory chain complex deficiency could be demonstrated in cultured fibroblasts or in mitochondria isolated from a muscle biopsy performed on the boy. In muscle isolated mitochondria, a progressive decrease of the rate of glutamate oxidation was observed after ADP addition; the rate of 2-ketoglutarate oxidation was low in the absence of ADP and did not increase after ADP addition. 2-KGD deficiency was demonstrated in fibroblasts from both children and in the boy's muscle and myoblasts. The 2-KGD complex is composed of three separate enzymes: E1, E2 and E3. We could demonstrate in our patient that the E1 and E3 subunits were normal, suggesting that the E2 component could be responsible for the defect.
Isolated rat liver mitochondria were split into three fractions of increasing density when applied to a Percoll gradient. NADH-ubiquinone oxidoreductase, succinate dehydrogenase and cytochrome-c oxidase but not F,-ATPase activities increased with density as well as respiratory rate in state 3 and the respiratory control index. Flow cytometry of mitochondrial density fractions stained with rhodaminerevealed the occurrence in each density fraction of two distinct mitochondrial populations with different fluorescence intensity. The high fluorescence population was minor and its proportion decreased with density. The extent of high fluorescence population staining depended on the deenergized state of the mitochondria suggesting that this population represents an immature form of the mitochondria which may develop into a fully functional organelle by the incorporation of structural and/or functional proteins.
Mitochondria play an essential role in the generation of the energy needed for eukaryotic cell life and in the release of molecules involved in initiation of cell death. Here we review the changes in isolated mitochondrial fluorescent populations as distinguished by flow cytometry during postnatal development and their regulation by thyroid hormones and catecholamines. The use of flow cytometry in the study of mitochondrial changes occurring under hypothyroidism, alcohol abuse and aging is also reviewed. ß
Monoclonal antibodies reacting with the inhibitor protein (IF1) of the mitochondrial ATPase/ATP synthase complex did not modify the IF1‐induced inhibition of soluble F1 ATPase activity. On the contrary, they increased the ATPase activity of inverted electron‐transport particles without inducing a significant release of IF1 from these particles. This suggested that IF1 could be linked to a membrane protein when it was not inhibiting the ATPase activity.
IF1 antibodies have been used to show that IF1 can bind not only to the β subunit of F1‐ATPase [Klein, G., Satre, M., Dianoux, A. C. & Vignais, P. V. (1981) Biochemistry 20, 1339–1344] but also to a protein present in the inner‐mitochondrial membrane. The cross‐linking of IF1 to this membrane protein gave a product of Mr 15000–16000 that migrated differently from IF1 and from the dimer of IF1 using SDS/PAGE. When the cross‐linked product was obtained by using a cleavable cross‐linking reagent, the complex between IF1 and the docking protein was partly dissociated and free IF1 was recovered. Considering the molecular mass of IF1, this docking protein for IF1 has apparent Mr 5000–6000.
The complex between IF1 and this receptor protein can be detected in low amounts by antibodies against IF1 in the absence of cross‐linking reagent. Since this complex remained in the pellet after treatment of the membrane with Triton X‐100, it should be a membrane protein. Therefore, IF1 can bind not only to its inhibitory‐binding site at the β subunit of F1, but also to a non inhibitory site which is a membrane protein of approximate Mr 5000–6000.
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