524 11. LOCALIZATION OF THE SITE OF INHIBITION 



P^ (inorganic phosi^hate) into cells without a corresponding depression of 

 respiration is, of course, not evidence for uncoupling. Oxidative phosphory- 

 lation proceeds normally without appreciable exchange of Pj between the 

 cell and the medium, so that when the classic uncouplers, such as 2,4-di- 

 nitrophenol, act upon cells there is no reduction in phosphate uptake un- 

 less growth is inhibited. Furthermore, in some cells the uptake of P^ is 

 regulated or metabolically dependent, so that an inhibitor could depress 

 the inward movement of Pj by a mechanism unrelated to the processes 

 of oxidative phosphorylation. Another criterion that has been used to 

 demonstrate uncoupling in cells is the rise in P, and fall in ATP produced 

 by a substance. This is certainly suggestive of uncoupling but does not 

 establish it. The levels of P^ and ATP within a cell depend on many reac- 

 tions and not solely on the rate of oxidative phosphorylation. An increased 

 utilization or enzymic splitting of ATP may lead to a new steady state in 

 which the ATP is lower and the P, higher than originally, if the rate of 

 ATP formation is not adequate to maintain the level. Yet another criterion 

 that has been suggested for uncoupling is a stimulation of respiration on 

 the basis that the rate of oxidation is limited by the phosphorylative reac- 

 tion. However, the dependency of the oxidative rate on phosphorylation 

 varies with the type of cell, the state of the cell, and the nature of the oxi- 

 dative pathway. A very active tissue utilizing ATP at a high rate may be 

 operating at maximal oxidative capacity and no stimulation would result 

 from uncoupling, whereas in resting cells, in which respiration is limited 

 by phosphate acceptors, a stimulation from uncoupling may be elicited. 

 Uncoupling agents also are able to exert an inhibitory effect on electron 

 transport and this may mask any stimulating action due to uncoupling. 

 Finally, an increase in respiration can be brought about in a variety of 

 ways unassociated with oxidative phosphorylation. These few conside- 

 rations indicate the difficulties that may be experienced in attempting to 

 establish uncoupling in living cells. 



The conclusive proof for an uncoupling site must therefore be derived 

 from experiments on units isolated from cells. These units are of some 

 complexity since as yet no single enzyme carrying out oxidative phospho- 

 rylation has been obtained. Mitochondria have been most commonly used 

 but submitochondrial particles obtained by various methods, such as son- 

 ic disintegration, are available and perhaps preferable. The results are 

 usually expressed in terms of the P : ratio, which is the number of phos- 

 phate groups esterified for each atom of oxygen reduced. It is obvious that 

 if the organic phosphates formed are split by phosphatases or react in any 

 way so that P, is set free, the experimental value of the P : ratio will 

 be lower than the true value. The phosphate esterified is usually determined 

 from (a) disappearance of P^ from the medium, (6) formation of ATP, 

 (c) formation of glucose-6-phosphate in the presence of hexokinase, or (d) 



