Control Points in Phosphorylating Respiration 133 



energy linkage in the pre-existing CARRIERqx. ^' X. The 

 scheme also shows that ADP is a necessary reaction partner 

 for incorporation of ^^Pj. On the other hand, it accounts for 

 the finding that reversible incorporation of ADP may proceed 

 in the absence of phosphate and that the incorporation of ^^Pj 

 may be inactivated by ageing [elimination of reaction (2)] 

 leaving reaction (3) intact. It is concluded that the terminal 

 reaction of oxidative phosphorylation has the form of reaction 

 (3), where P /^ X is a bifunctional phosphate-transferring 

 enzyme. The findings exclude an alternative formulation 

 involving an ADP '^ X complex (Wadkins and Lehninger, 

 1959). 



The experiments on the exchange reactions of ATP thus 

 establish the sequence of entry of orthophosphate and ADP 

 in the mechanism of energy coupling shown in reactions (1), 

 (2) and (3). These findings are given additional support by 

 the following experiments. 



Site of action of uncoupling agents 



Measurement of the effects of a variety of different un- 

 coupling agents on the rate of the two ATP exchanges, the 

 P/0 ratio during oxidation of p-hydroxybutyrate, as well as 

 the ATPase activity (Cooper and Lehninger, 1957a) [a reflec- 

 tion of the reverse of reactions (7) and (6), followed presumably 

 by hydrolysis of carrier ^' X or its energetic equivalent] 

 yielded the data summarized in Table I. Freshly prepared 

 digit onin subfragments were studied. 



The three agents DNP, dicoumarol and gramicidin uncouple 

 oxidative phosphorylation, inhibit both exchange reactions of 

 ATP and stimulate ATPase activity, demonstrating that they 

 act on a component common to all the partial reactions of 

 oxidative phosphorylation. 



Thyroxine and its analogues, and also Ca^+j although potent 

 uncoupling agents in intact mitochondria, do not uncouple 

 oxidative phosphorylation in the digitonin fragments, nor 

 do they affect any of the partial reactions at concentrations 

 which uncouple in intact mitochondria. They have a mode of 



