Coupling of Adenosine Triphosphate Formation in Mitochondria to the Formation of Nucleoside Triphosphates: INVOLVEMENT OF NUCLEOSIDE DIPHOSPHOKINASE

Abstract

Abstract Addition of the nucleoside diphosphates CDP, UDP, TDP, and IDP to intact, respiring, rat liver mitochondria immediately following ATP formation results in a marked stimulation of respiration at all concentrations of nucleotide tested. Addition of GDP stimulates respiration when added at concentrations below 150 µm, whereas at higher concentrations this nucleotide has much less effect on respiration and prevents stimulation given by other nucleoside diphosphates. Half-maximal stimulation of respiration is provided by less than 150 µm of each nucleotide. Nucleoside triphosphate formation is associated directly with stimulation of respiration by nucleoside diphosphates. CTP, UTP, TTP, and ITP are formed in good yield at all concentrations of nucleoside diphosphate tested, whereas GTP is formed in best yield at concentrations of GDP below 150 µm. Concentrations of nucleoside diphosphates which give half-maximal rates of nucleoside triphosphate formation correlate well with concentrations necessary to give half-maximal stimulation of respiration. Concentrations of GDP higher than 150 µm inhibit both the formation of GTP and the formation of other nucleoside triphosphates. Oligomycin and 2,4-dinitrophenol, inhibitors of oxidative phosphorylation, also inhibit nucleoside triphosphate formation. Partial removal of nucleoside diphosphokinase localized in the outer mitochondrial compartment markedly reduces both nucleoside diphosphate stimulation of respiration and nucleoside triphosphate formation. Kinetic analysis of the solubilized preparation reveals typical Michaelis-Menten kinetics for the substrate pairs ATP-CDP, ATP-UDP, and ATP-TDP, a slight deviation from normal kinetic behavior for the substrate pair ATP-IDP, and a marked deviation from normal behavior for the substrate pair ATP-GDP. GDP is a potent inhibitor of the enzyme at concentrations exceeding 150 µm and, as a result, biphasic kinetic response curves are obtained when the substrate pair is ATP-GDP. Taken together these results show that ATP formation in intact rat liver mitochondria can be readily coupled to nucleoside triphosphate formation when nucleoside diphosphates are supplied exogenously, that nucleoside diphosphokinase localized in the outer mitochondrial compartment is intimately associated with the coupling process, and that GDP via its action on nucleoside diphosphokinase is a potent inhibitor of nucleoside triphosphate formation.