GENERAL DEVELOPMEXT OF A LOCALIZATION PROGRAM 529 



The Sequence of Experimental Localization 



It is impossible to present any general procedure to follow in all cases 

 because the situations presented by various inhibitors will often have 

 little in common. Some frequently occurring situations will be discussed 

 briefly to em^jhasize the necessity for a more complete analysis than is 

 usually found in the literature. This approach is particularly to be contras- 

 ted to the common type of report wherein a single positive result with an 

 inhibitor is used as the basis for a designation of the site of action. An 

 inhibitor that has been found to depress respiration is immediately tested 

 on the tricarboxylic acid cycle and if inhibition is found, whatever the de- 

 gree, the cycle is stated to be the site of action; such inferences are invalid 

 in general and tenuous under the most favorable circumstances. A few 

 comments will now be made on the suggested approach as applied to the 

 localization of inhibitors depressing respiration or cell growth. 



A depression of respiration by a substance may be observed initially in 

 a variety of ways. Let us assume, for generality, that a substance has been 

 found to reduce the respiration of an intact organ as measured by changes 

 in the arteriovenous oxygen difference, that a dosage or concentration 

 curve has been obtained, that the inhibition has been shown to be readily 

 reversible, and that the nature of the inhibitor gives no clue as to its site 

 of action. What are some of the j)ossible mechanisms by which this 

 respiratory depression could be brought about? 



I. Inhibition of some step in electron transport that is common to most 

 oxidation systems (such as one of the cytochromes). 



II. Inhibition of an enzyme in the tricarboxylic acid cycle (such as 

 isocitric dehydrogenase or aconitase) or of an enzyme introducing a sub- 

 stance into the cycle (such as the condensing enzyme or pyruvate oxidase). 



III. Inhibition of a i)athway leading to the tricarboxylic acid cycle 

 (such as glycolysis if glucose or glycogen are the principal substrates or 

 the fatty acid breakdown helix if fatty acids are important substrates). 



IV. Decrease in permeability or the activity of a transport system for 

 an exogenous substrate. 



V. Primary depression of functional activity of the tissue leading to 

 reduction of the respiration. 



Although the major possibilities listed are few, a survey of the commonly 

 used inhibitors that depress respiration will show that almost all act in 

 one of these ways. It is unnecessary initially to worry about more esoteric 

 mechanisms. 



