Reconstitution of the Dinitrophenol-Sensitive Atp-Adp Exchange Reaction of Oxidative Phosphorylation

Abstract

Intact rat liver mitochondrial as well as fragments of mitochondrial membranes capable of oxidative phosphorylation2 catalyze an exchange reaction between ATP and ADP which is inhibited by 2,4-dinitrophenol (DNP). The exchange enzyme has been postulated to catalyze the terminal reaction of oxidative phosphorylation by which ATP is formed.'-5 The ATP-ADP exchange reaction is inhibited by dinitrophenol only in freshly prepared particles capable of phosphorylation; aging of the particles causes no loss of activity of the exchange but results in loss of its DNP-sensitivity.2 Similarly, azide has no inhibitory effect on the ATP-ADP exchange, but abolishes its sensitivity to DNP.2 The stability of the ATP-ADP exchange enzyme made possible its extraction in soluble form2 and its purification to a high degree.6 The soluble form of the enzyme is completely insensitive to DNP.2,6These findings thus show that DNP-sensitivity is not intrinsic but is conferred on the ATP-ADP exchange in fresh mitochondria or membrane fragments because it is in functional equilibrium with an earlier enzymatic reaction of the respiratory energy coupling mechanism which is sensitive to DNP, from which it can be dissociated by aging, azide, or physical separation. This paper describes the restoration of DNP-sensitivity to the soluble, DNPinsensitive ATP-ADP exchange enzyme separated from rat liver mitochondria, by recombining it with mitochondrial membrane fragments prepared by the action of digitonin.7 Such "recombination," which is specific, indicates that the soluble ATP-ADP exchange enzyme we have isolated is a participant in the mechanism of respiratory energy-coupling and differentiates it from other mitochondrial enzymes catalyzing ATP-ADP exchanges2 which are not relevant to oxidative phosphorylation.Methods.-The ATP-ADP exchange rate was measured with p32-or C'4-labeled ADP using the paper chromatographic separation described before.2 Initial incorporation rates under conditions of substrate saturation were measured. Phosphorylating digitonin particles from rat liver mitochondria were prepared according to Devlin and Lehninger.7 Soluble ATP-ADP exchange enzyme was prepared according to Wadkins and Lehninger.2 The purification of the ATP-ADP ex-

Phosphorylation by Barley Root Mitochondria & Phosphate Absorption by Barley Roots

“…This results in the observed decrease in Vmax through a decrease in turnover which is directly proportional to the rate of electron flow. Azide also inhibits the ATP-P1 exchange reaction of oxidative phosphorylation (reactions b & c) but the ATP-ADP exchange (reaction c) is relatively insensitive (22,23 (21) found that ammonia inhibits the oxidation of DPNH in beet root mitochondria. They suggest that this is the mechanism of the inhibition of respiration by ammonia in intact plants.…”