Respiration of mitochondria in living organisms is controlled by the ATP/ADP ratio and phosphorylation pattern of cytochrome c oxidase
The "second mechanism of respiratory control" (allosteric ATP-inhibition of cytochrome c oxidase (CcO)) is demonstrated for the first time in intact isolated rat liver and heart mitochondria. The problems of measuring the kinetics of allosteric ATP-inhibition in isolated mitochondria we...
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|Zusammenfassung:||The "second mechanism of respiratory control" (allosteric ATP-inhibition of cytochrome c oxidase (CcO)) is demonstrated for the first time in intact isolated rat liver and heart mitochondria. The problems of measuring the kinetics of allosteric ATP-inhibition in isolated mitochondria were investigated. And it was found that only at very high ATP/ADP ratios, this inhibition is obtained and requires an ATP-regenerating system consisting of phosphoenolpyruvate (PEP) and pyruvate kinase (PK). The allosteric ATP-inhibition can be switched off probably by dephosphorylation of a serine at CcO subunit-I. The phosphorylation of CcO at serine, threonine and tyrosine was studied in isolated mitochondria by extracting complex IV of the respiratory chain (CcO) by BN-PAGE (blue-nativepolyacrylamide- gel-electrophoresis), SDS-PAGE and Western blotting with the corresponding antibodies against the phosphorylated amino acids. The extent of allosteric ATP-inhibition of CcO varied in different preparations of mitochondria, which was suggested to be based on the psychological situation of the animal at the time of killing. Incubation of bovine heart tissue slices with insulin decreased the allosteric ATP-inhibition and phosphorylation of CcO subunit-I at serine. By using a TPP+ (tetraphenylphosphonium)- electrode, and in collaboration with Katrin Staniek, Veterinary University, Vienna, a reversible decrease of the mitochondrial membrane potential (DYm) by PEP and PK through the DYm-independent allosteric ATP-inhibition of CcO at high ATP/ADP ratios was demonstrated in isolated rat liver mitochondria for the first time. It is proposed that respiration in living eukaryotic organisms is normally regulated by the DYm-independent ‘allosteric ATPinhibition of CcO’, and only when the allosteric ATP-inhibition is switched off under stress, respiration is regulated by ‘respiratory control’, based on DYm according to the Mitchell Theory.|