136 Albert L. Lehninger, et al. 



that DNP causes some limitation on, or removal of, P /^ X 

 by an action on either reaction (1) or reaction (2) in fresh 

 preparations. Ageing thus causes inactivation of a reaction 

 located between the site of action of DNP and the ATP-ADP 

 exchange. It is clear also that the effect of ageing is not the 

 equivalent of the action of DNP, as has been suggested by- 

 others, since ageing causes loss of ATPase activity of the 

 mitochondrial fragments whereas DNP stimulates it. 



In similar experiments it has been found that sensitivity of 

 the ATP-ADP exchange to dicoumarol is similarly abolished 

 by ageing, suggesting that it has a site of action near that of 

 DNP, but not necessarily at the same point. 



A third type of experiment has confirmed these implications 

 and made it possible to establish the approximate sites of 

 action of azide and of high concentrations of neutral salts and 

 sucrose, which are agencies causing uncoupling and inhibition 

 of the ATP-32P. exchange and ATPase. Data in Table I show 

 azide to be an uncoupling agent and an inhibitor of the ATP- 

 32p. exchange but that it inhibits ATPase activity, and has 

 no effect on the ATP-ADP exchange, indicating that it has a 

 site of action different from that of DNP. This was not only 

 confirmed, but the approximate site of action of azide was 

 defined by the experiments summarized in Table II. It is seen 

 that azide, although not inhibitory to the ATP-ADP exchange, 

 aboHshes the sensitivity of the ATP-ADP exchange to DNP 

 and also to dicoumarol in fresh submitochondrial fragments. 

 Therefore azide has a site of action which lies between the site 

 of action of DNP and the terminal ATP-ADP exchange. 

 Similarly, azide was found to abolish the stimulating effect of 

 DNP on ATPase activity, an observation wholly consistent 

 with this conclusion (Wadkins and Lehninger, 1959). It is 

 possible that azide inhibits a phase of reaction (2) which is also 

 easily inactivated by ageing. 



Similar experiments have shown that DNP-sensitivity of 

 the ATP-ADP exchange, as well as DNP-stimulation of ATP- 

 ase activity, is prevented by -3 m KCl and by -5 m sucrose. 

 Clearly, these agents must also act at or near the site of action 



