Studies on the relation of the adenosinetriphosphate-inorganic phosphate exchange reaction of mitochondria to oxidative phosphorylation

Abstract

The effects of a number of various agents on the inorganic phosphate-adenosinetriphosphate exchange (P i-ATP exchange) reaction catalyzed by rat-liver mitochondria have been studied. The types of agents included certain dyes, various respiratory inhibitors, and a number of other compounds of known interest in studies of oxidative phosphorylation. Oxidized, but not reduced, 2,6-dichlorophenolindophenol completely inhibited P i-ATP exchange and oxidative phosphorylation and partially depressed respiration supported by various substrates. The effect on the respiration was not eliminated by 2,4-dinitrophenol. Reduction of the dye after previous treatment of the mitochondria with the oxidized form partially restored the P i-ATP exchange. Methylene blue acted in all respects similarly to 2,6-dichlorophenolindophenol, while ferricyanide and a number of other one-electron acceptors were without inhibitory effects. Cyanide and antimycin A inhibited P i-ATP exchange to a maximal extent of 20–40%, whereas amytal gave an inhibition which was progressive with increasing concentration and could approach completeness. The progressive character of the amytal inhibition was not eliminated by the presence of antimycin A. The effect of amytal, in contrast to those of cyanide and antimycin A, was partially prevented by succinate; antimycin A, but not cyanide, abolished the succinate effect. The effects of adenosinemono- and diphosphate, p-chloromercuribenzoate, azide, fluoride, arsenate, ethylenediaminetetraacetate, deoxycholate and 2,4-dinitrophenol on P i-ATP exchange were investigated and compared with those on oxidative phosphorylation and/or mitochondrial adenosinetriphosphatase reactions. A short exposure of mitochondria to 2,4-dinitrophenol at 2°C resulted in a depression of the P i-ATP exchange with a maintenance of the net phosphate uptake upon the addition of substrate. On the basis of the data obtained it is concluded that the P i-ATP exchange reaction does not reflect all three phosphorylations occurring along the respiratory chain in a random manner, but is related mainly to one of these: that occurring at the diaphorase level. The implications of these conclusions for the mechanisms of respiratory chain phosphorylations and of dinitrophenol-activated adenosinetriphosphatase is discussed.