The Regulation of Nicotinamide Adenine Dinucleotide-linked Substrate Oxidation in Isolated Liver Mitochondria

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Abstract A mechanism for regulating the rate of NAD-linked substrate oxidation in isolated liver mitochondria was investigated. Addition of oligomycin and α-ketoglutarate to isolated uncoupled rat liver mitochondria oxidizing an NAD-linked substrate such as l(-)-palmitylcarnitine plus l-malate caused an abrupt inhibition of oxygen consumption and of citrate, acetoacetate, and β-hydroxybutyrate synthesis. This respiratory inhibition was nonspecific for NAD-linked substrates and was not observed with flavin-linked substrates such as succinate. The molecular species which was responsible for the inhibition of NAD-linked substrate oxidation was established as guanosine triphosphate by four types of experiments: (a) the intramitochondrial GTP level increased coincident with the establishment of the inhibited state; (b) the induction of the inhibitory state was prevented, or released, or both, upon the inclusion of exogenous ADP in the mitochondrial incubation, presumably due to the reaction of ADP and GTP with the nucleoside diphosphokinase forming ATP and GDP; (c) the establishment of the respiratory inhibition was not observed in guinea pig liver mitochondria in the presence of a source of oxalacetate for phosphoenolpyruvate synthesis by the GTP-requiring phosphoenolpyruvate carboxykinase; and (d) addition of phosphoenolpyruvate to reverse the phosphoenolpyruvate carboxykinase reaction resulted in the synthesis of GTP and caused an abrupt inhibition of l(-)-palmitylcarnitine oxidation in isolated guinea pig liver mitochondria. This respiratory inhibition by phosphoenolpyruvate addition was prevented by including l-malate or ADP in the incubation mixture. The experiments described in this communication suggest that the intramitochondrial GTP level may be a potent regulator of the flux of reducing equivalents derived from the primary NAD-linked dehydrogenases into the electron transfer chain and the consequential generation of ATP in the oxidative phosphorylation sequence of the mitochondrion.