526 11. LOCALIZATION OF THE SITE OF INHIBITION 



to be sensitive to structural changes, for example, and any agent that 

 induced disruption of the organized structure could uncouple secondarily. 

 The relationship between mitochondrial swelling and uncoupling is still 

 obscure but in any event swelling probably means an alteration of the in- 

 tramitochondrial state. Ionic changes, fluctuations in pH, or the release 

 of natural uncoupling agents might mediate the effect. The use of submito- 

 chondrial particles might eliminate these problems. 



A generalized oxidative phosphorylation system may be represented as: 



(11-6) 



A„ 



^X - P- 



_ LadpJ 



kY - P 



where A is any substrate or coenzyme, X represents all the possible com- 

 ponents that may be involved in phosphate transfer to ADP, and Y rep- 

 resents any substance that is used to trap the phosphate in the test sys- 

 tem. Three basically different types of uncoupling mechanism are possible. 



(1) The uncoupling agent is able to accept the phosphate from X-P, com- 

 peting with ADP, but, being unstable, breaks down rapidly so that little 

 or no ATP is formed and the P, concentration does not change appreciably. 



(2) The uncoupling agent is able to replace the phosphate so that complexes 

 such as X-I, ADP-I, or Y-I may be formed, where I is the inhibitor or 

 uncoupler. It may be that one of these complexes is unstable and the I 

 acceptor is regenerated but in either case there is a reduction of the phos- 

 phorylation. (3) The nncoupling agent in some manner (without partici- 

 pating in the reactions) prevents the transfer of Pj to ADP, either by allow- 

 ing oxidation to proceed without any reactions involving phosphate, or 

 by inducing instability into the X-P complex, or by inhibiting the transfer 

 from X-P to ADP. Because knowledge of the intimate details of phospho- 

 rylative coupling is lacking, it is impossil)le at the present time to loca- 

 lize accurately uncouplers of the third type. It is often not too easy to 

 distinguish between the third type and the other two mechanisms unless 

 the abnormal complexes have lifetimes sufficiently long so that they may 

 be demonstrated. 



A definition of uncoupling agents may now be projjosed on the basis of 

 these observations. An uncoupling agent interferes directly in one of the 

 reactions between oxidation and the formation of ATP, with the result that 

 the obligatory coupling is abolished and oxidation may proceed without 

 normal ATP synthesis. The definition adopted here implies that the sub- 

 stance acts in some manner between A^^^-^A^a- and ADP -^ ATP in 

 scheme 11-6. Substances that act indirectly upon these reactions or that 

 affect systems peripheral to the phosphorylation process act fundamentally 

 in a different way and should not be classified as uncouplers. Evidence for 

 an uncoupling site of action for a substance must therefore include a dem- 



