METHODS OF LOCALIZATION 525 



formation of creatine phosphate in the presence of creatine phosphokinase. 

 When trapping systems for the esterified phosphate are used, it must be 

 remembered that an inhibitor can also act on the transferring enzymes. 

 For example, when the ATP formed in oxidative phosphorylation is uti- 

 lized in the formation of glucose-6-phosphate from glucose, an inhibition 

 of the hexokinase will depress the formation of the glucose -6-phosphate 

 and the ATP synthesized may either accumulate or a greater fraction be 

 eventually broken down by ATPase, the experimental P : ratio decreas- 

 ing. Also, as in cells, an activation of ATPase by the inhibitor, either 

 directly or indirectly, will tend to reduce the experimental P : ratio.* 

 Difficulties in interpretation sometimes arise even in mitochondrial stud- 

 ies. There is the possibility of phosphorylating and nonphosphorylating 

 pathways and a shift in the relative activities of these. In such a system, 



V \^ 



A O2 



C 



/ 

 B 



the experimentally determined P : ratio will in general not accurately 

 represent the phosphorylative efficiency of the A -> B ^ O2 pathway. 

 Furthermore, any variation in the oxygen reduced by the A ^ C -> O2 

 or B -» C ^ 0-2 pathways will alter the P : ratio. Thus the stimulation 

 of a nonphosphorylating pathway will lower the P : ratio. Whether this 

 should be termed uncoupling or not is sometimes difficult to decide. When 

 the nonphosphorylating pathway is unrelated to the phosphorylating path- 

 way, i.e., with a different substrate or involving different electron transport 

 systems, the eifect is not true uncoupling in that no change in the oxidative 

 phosphorylation processes has occurred, but there are instances where the 

 same substrate is involved and it is not certain if a new transport pathway 

 has been opened up or the old pathway uncoupled. 



A further difficulty, which is actually inherent in much work with inhi- 

 bitors, exists in the demonstration that the uncoupling, if it does occur, 

 is the result of a direct action of the substance on oxidative phosphoryl- 

 ation or whether it is due to the disturbances created by the substance 

 acting elsewhere. Mitochondrial oxidative phosphorylation would appear 



* If tlie ATPase activated is not a component of the oxidative phosphorylation 

 pathway, there is httle justification for concluding that a drop in the P : O ratio is 

 indicative of uncouphng. However, if the ATPase activity measured is an expression 

 of the reversal of the phospliorylation process and involves the same enzymic system, 

 the increase in the ATPase activity will implicate an uncoupling mechanism. 



