Control Points in Phosphorylating Respiration 131 



The reaction is inhibited by 2 : 4-dinitrophenol (DNP) and 

 other uncouphng agents with about the same sensitivity as 

 oxidative phosphorylation. The rate of this exchange reaction 

 is maximum when the respiratory carriers of the preparation 

 are maintained in the oxidized state (Wadkins and Lehninger, 

 1957). ADP has been demonstrated to be an essential com- 

 ponent of the ATP-^^Pi exchange reaction (Cooper and 

 Lehninger, 1957b). 



A second exchange reaction of ATP is catalysed by the 

 phosphorylating respiratory fragments, namely the ATP- 

 ADP exchange, in which ^^P- or ^^C-labelled ADP is incor- 

 porated bodily into ATP in a reversible manner in the absence 

 of net electron transport (Lehninger et al., 1958; Cooper and 

 Lehninger, 19576; Wadkins and Lehninger, 1958). The 

 ATP-ADP exchange is very rapid (faster than the ATP-^^p. 

 exchange) and proceeds to isotopic equilibrium. The ATP- 

 ADP exchange is also inhibited by DNP in freshly prepared 

 phosphorylating fragments, showing its relationship to oxida- 

 tive phosphorylation. The DNP-insensitive ATP-ADP ex- 

 change reactions associated with other phosphate-transferring 

 reactions, as in glutathione and glutamic synthetases, the 

 succinate-linked P-enzyme, etc. are absent from the fragment 

 preparations. The ATP-ADP exchange of oxidative phos- 

 phorylation requires Mg2+ but no other dialysable component. 



The important observation was made that inorganic phos- 

 phate is not required for the ATP-ADP exchange nor does it 

 affect the rate of the exchange over a 5000-fold range of con- 

 centration (Lehninger, et ah, 1958; Wadkins and Lehninger, 

 1958), suggesting that inorganic phosphate is taken up in 

 another reaction, probably preceding the uptake of ADP. 

 This suggestion was given very firm support by the finding 

 that ageing of the enzyme fragments at 2° for 48 hours or 

 longer caused complete loss of the ability to catalyse the 

 incorporation of ^^p. j^to ATP, but resulted in no loss of 

 activity of the ATP-ADP exchange. Thus, inorganic phos- 

 phate must be incorporated by a rather labile reaction which 

 precedes the entry of ADP. 



