ABSTRACT
Redistribution potentiometric dyes represent a powerful tool for monitoring membrane potential of mitochondria, especially when these dyes are used with flow cytometry. In particular, tetramethylrhodamine methyl ester proved to be suitable for the screening of mitochondrial membrane potential in cultured human skin fibroblasts from patients suffering from different defects of oxidative phosphorylation. We have developed a method that makes it possible to measure the changes in mitochondrial membrane potential, or to assess the differences between respective mitochondrial membrane potentials in investigated cells and controls in the absolute scale of millivolts. Our approach employs the fact that a logarithmic transformation of Nernst equation-controlled intensity of fluorescence from potentiometric dyes accumulated in mitochondria leads to a linear scale for mitochondrial membrane potentials.
Subject(s)
Membrane Potentials , Mitochondria, Liver/metabolism , Adenosine Diphosphate/pharmacology , Aldehydes/pharmacology , Animals , Calibration , Cells, Cultured , Fibroblasts/cytology , Flow Cytometry , Fluorescence , Humans , Membrane Potentials/drug effects , Mitochondria, Liver/drug effects , RatsABSTRACT
A maternally inherited and practically homoplasmic mitochondrial (mtDNA) mutation, 8527A>G, changing the initiation codon AUG into GUG, normally coding for a valine, was observed in the ATP6 gene encoding the ATPase subunit a. No alternate Met codon could replace the normal translational initiator. The patient harboring this mutation exhibited clinical symptoms suggesting a mitochondrial disease but his mother who carried the same mtDNA mutation was healthy. The mutation was absent from 100 controls and occurred once amongst 44 patients suspected of Leber Hereditary Optic Neuropathy (LHON) but devoid of typical LHON mutations. In patient fibroblasts, no effect of 8527A>G mutation could be demonstrated on the biosynthesis of mtDNA-encoded proteins, on size and the content of ATPase subunit a, on ATP hydrolysis and on mitochondrial membrane potential. In addition, ATP synthesis was barely decreased. Therefore, GUG is a functional initiation codon for the human ATP6 gene.
Subject(s)
Adenosine Triphosphatases/genetics , Codon, Initiator , Mitochondria/metabolism , Protein Biosynthesis , Adenosine Triphosphatases/chemistry , Adenosine Triphosphate/biosynthesis , Adenosine Triphosphate/chemistry , Adult , Blotting, Western , Child , DNA/metabolism , Fibroblasts/metabolism , Flow Cytometry , Humans , Male , Mitochondrial Proton-Translocating ATPases , Muscles/metabolism , Mutation , Oxygen/metabolism , Phosphorylation , Skin/metabolism , Valine/geneticsABSTRACT
Glycerophosphate (GP)-dependent, ferricyanide-induced hydrogen peroxide production was studied in brown adipose tissue mitochondria from newborn rats. Relations between the rate of hydrogen peroxide production and total amount of hydrogen peroxide produced at different GP and ferricyanide concentrations were determined. It was found that the rate of hydrogen peroxide production increases with increasing GP concentration and decreases with increasing ferricyanide concentration. Total amount of hydrogen peroxide produced increases with increasing ferricyanide concentration, however, not proportionally, and the efficiency of this process (oxygen/ferricyanide ratio) strongly declines. Data presented provide further information on the character and kinetics of hydrogen peroxide production by mammalian mitochondrial glycerophosphate dehydrogenase.
