Control Points in Phosphorylating Respiration 135 



A second type of experiment made it possible to pinpoint 

 more closely the site of action of different uncoupling agents 

 in the coupling mechanism. Ageing of the submitochondrial 

 fragments at 2° for 72 hours has been found to cause complete 

 loss of coupling activity, complete loss of ATP-^^p. exchange 

 activity, complete loss of ATPase, but relatively insignificant 

 loss in the activity of the ATP-ADP exchange, indicating that 

 reaction (7) can proceed independently of the other partial 

 reactions of coupling (Wadkins and Lehninger, 1959). 



100 



50 - 



A 



TOTAL ACTIVITY 



\ 



^^^DNP-RESISTANT 

 ° ACTIVITY 



ATP-ADP CC) EXCHANGE 



24 48 72 



AGE OF ENZYME AT 2»( HOURS) 



Fig. 1. Loss of sensitivity of the 

 ATP-ADP exchange to DNP on 

 ageing of phosphorylating frag- 

 ments. 



However, the very important finding was made that such 

 ageing of the particles, although causing no significant loss of 

 ATP-ADP exchange activity, does cause loss of the sensitivity 

 of this reaction to inhibition by DNP and dicoumarol, the 

 rate of loss of the sensitivity being exactly parallel to the rate 

 of inactivation of the ATP-^^Pj exchange (Fig. 1) (Wadkins 

 and Lehninger, 1959). This finding clearly indicates that the 

 ATP-ADP exchange per se is inherently insensitive to inhibi- 

 tion by DNP and that DNP-sensitivity is conferred on it by a 

 factor which is labile to ageing. Since the ATP-ADP exchange 

 has a reaction component (P '--' X) which is shared with the 

 preceding reaction (2) in the hypothesis, it appears probable 



