Efflux of Adenine Nucleotides from Rat Liver Mitochondria

A rapid and selective efflux of 75 to 80% of the intramitochondrial adenine nucleotides (AdN) has been obtained by incubation with 5 mm Mg++ and 10 mm inorganic phosphate in the presence of 1 mm ethylenediaminetetraacetate, with little or no decrease in the P:O ratio, active respiratory state, or size of the pyridine nucleotide pool. The initial rate of AdN efflux is 5 nmoles per mg of protein per min at 30°. The efflux is a linear function of the [Pi] but with increasing [Mg++] exhibits an apparent Km for Mg++ of 0.6 mm with mitochondria incubated in the presence of 0.1 mm EDTA. The rate of efflux of AdN is considerably reduced by respiratory inhibitors and uncouplers, including rotenone-malonate, dinitrophenol, arsenate, and oligomycin, or by an activator of ion transport such as valinomycin. All of these agents cause an increase in the proportion of AMP or ADP, or both, within the mitochondrion. Pi competitively reverses the inhibition of AdN efflux by arsenate and concomitantly increases the proportion of mitochondrial ATP. The Mg++-Pi-induced efflux of AdN can be prevented by adding sufficient exogenous ADP to maintain an intra- to extramitochondrial AdN ratio of about 20. The omission of Mg++ or replacement by 1 mm Ca++ has little effect on the rate of AdN efflux, but lowers markedly the intra- to extramitochondrial AdN equilibrium ratio to 3.1 and 1.6, respectively. Addition of atractyloside to Mg++- and Pi-treated mitochondria completely reverses the inhibitory effect of exogenous ADP on the efflux of AdN, but is without effect on the Ca++-treated mitochondria. It is concluded that the efflux of AdN from rat liver mitochondria under atractyloside sensitive conditions is associated with the AdN translocation site and is selective for ATP, while AMP and probably ADP are inert. The mechanism of action of Pi, Mg++, and EDTA on AdN efflux is presented in terms of their effect on ion transport and mitochondrial integrity.