34 ALBERT L. LEHNINGER 



group of ATP exchanges very rapidly, a reaction which is completely 

 inhibited by dinitrophenol [12]. 



Both the ATP-ase and ATP-F;^'- exchange have been studied to 

 greatest advantage in so-called digitonin fragments of the membranes of 

 rat liver mitochondria [2, 13], which contain complete respiratory chains 

 and coupling mechanisms but do not show Krebs cycle activity. These 

 fragments are relatively free of enzymes not relevant to oxidative phos- 

 phorylation and are not so subject to compartmentation phenomena as 

 are intact mitochondria. With these fragments it was found that the 

 partial reactions are specific for nucleotides of adenine. Further, the require- 

 ments and kinetics of the ATP exchange reaction could be examined more 

 closely. It was found that ADP was a necessary componerit in the ATP-Pj^^ 

 exchange [14] and also that this exchange was most rapid when the 

 respiratory carriers were in the fully oxidized state [15]. 



During further examination of the mechanism of the ATP-P^^^ ex- 

 change, it was found that digitonin preparations also catalyze an exchange 

 of labelled ADP into ATP which was inhibited by DNP [14, 16]. This 

 exchange, which is specific for adenine nucleotides, does not require in- 

 organic phosphate and was found not to be caused by other phosphate- 

 transferring enzymes known to catalyze ATP-ADP exchanges, such as 

 adenylate kinase and protein phosphokinase. The exchange activity is 

 stable but on ageing loses its sensitivity to DNP. This striking finding was 

 corroborated by independent experiments with azide; this agent does not 

 afiect the rate of the ATP-ADP exchange but prevents it from being 

 inhibited by DNP. The tentative conclusion was drawn that the ATP- 

 ADP exchange reaction is a reflection of the action of the terminal enzyme 

 of oxidative phosphorylation, but that this enzyme is not itself sensitive 

 to DNP. However, it was postulated that its sensitivity to DNP was 

 conferred on it because it is in equilibrium with a preceding reaction in 

 the coupling sequence which has a DNP-sensitive component. 



The information on the ATP-ase activity and the phosphate and ADP 

 exchange reactions therefore suggested that the general form of the 

 energy-coupling reactions could be expressed bv the following equations 

 [2, 14. 15]: 



Carrier r^ J + X — ^ Carrier^^ '--' X (i) 



Carrier^^j.'--' X + P^ ^ ^ Carrier^^ + P^—X (2) 

 P--X + ADP TZlATP + X (3) 



Reaction (3) thus accounts for the ATP-ADP exchange, reactions (2) 

 + (3) for the ATP-Pj-'^- exchange, and the sequence of reactions 3 + 2 

 plus the following reaction (4) for DNP-stimulated ATP-ase activity: 



Carrier '-^ X — ^-> Carrier + X (4) 



