METHODS OF LOCALIZATION 523 



Use of Reconstituted Enzyme Systems 



In those instances where an organized multienzyme complex can be 

 broken down into its component enzyme units, these individual units can 

 usually be tested directly for susceptibility to the inhibitor. However, 

 an even more potent method is available if the single enzymes can be put 

 back together in either partial or total reconstitution of the original sys- 

 tem. Certain parts of a linear sequence may be reformed and tested with the 

 inhibitor and occasionally artificial or unnatural substrates or intermediates 

 may be introduced. In this manner it is usually reasonably easy to locate 

 the enzyme attacked, providing it is a known and isolatable component. 

 This approach is best illustrated by the recent work of Keilin and King 

 (1960) on the sites of inhibition of several inhibitors of the succinate oxi- 

 dase system. This electron transport sequence was broken down into its 

 several known enzymes and reconstituted in different ways, and, in ad- 

 dition, electron donors and acceptors were introduced so that it was pos- 

 sible to test almost every individual unit or any partial sequence. 



Demonstration of an Uncoupling Site of Action 



When a substance is capable of producing a disturbance in cell function 

 or synthetic processes possibly attributable to an interference with energy 

 flow, and yet does not exhibit a significant depressant action on the respi- 

 ration in the same concentration range, it is usual to look for the site of 

 inhibition in the reactions whereby high-energy phosphorylations are 

 coupled with oxidations. Substances that dissociate oxidative phosphory- 

 lation have been called uncoupling agents or uncouplers. Such inhibitors 

 will be discussed in detail in a later chapter but it is appropriate here to 

 examine critically the evidence that may be presented for an uncoupling 

 site of action. The problem would be clarified if a rigorous and generally 

 acceptable definition of uncoupling existed but the literature contains sur- 

 prisingly few exact or carefully considered definitions of this phenomenon. 

 The best general discussion of uncoupling and the methods for demon- 

 strating it is perhaps the review by Brody (1955). A brief evaluation of 

 the preparations used in uncoupling studies will illustrate some of the pro- 

 blems in localization and may help to formulate a provisional definition. 



Preparations of cells or tissues are unsatisfactory for proving that a 

 substance uncouples oxidative phosphorylation because it is impossible 

 at the present time to determine accurately the rate of ATP formation 

 within the cell wdiere many metabolic reactions involving ATP proceed 

 simultaneously.* The observation that a substance reduces the uptake of 



* The immediate product of high-energy phosphorylation will be designated here 

 as ATP for simplicity, since this would appear to be generally the most important 

 substance. Other nucleotides or unknown acceptors, however, may play a role in the 

 conservation of oxidative energy. 



