528 11. LOCALIZATION OF THE SITE OF INHIBITION 



(2) The time relationships of the inhibition should be investigated and 

 this includes both the rate at which the inhibition is developed and the 

 degree of reversibility of the inhibition. A delayed onset of inhibition is 

 sometimes indicative of the metabolism of the substance to an active in- 

 hibitor or of a lethal synthesis. The failure of the inhibition to be reversed 

 readily when the substance is removed is also suggestive of mechanisms that 

 may facilitate the localization of the site of action. 



Planning the Program of Localization 



It is now of the utmost importance to map out a plan of attack. One 

 must do this on the basis of the data already obtained, the nature of the 

 inhibitory action, the system that has been found to be affected, and a 

 consideration of the properties of the inhibitor itself. It is well to gather 

 together all of the pertinent information on the physical and chemical 

 characteristics of the inhibitor. Such properties as solubilities, p^^'s, sta- 

 bility, possible isomerism, complexing abilities, chemical reactivity, and 

 special structural aspects can be crucial ])oints in either planning or in- 

 terpreting the work. Of course, the relationship of the compound, struc- 

 turally or in other ways, to naturally occurring substance^ or other inhi- 

 bitors whose actions are known will often suggest sites of action. In other 

 words, an attack can be made much more efficient and productive when 

 all the available data are at hand to guide it. 



Before outlining the initial phases of the localization, it is necessary 

 to consider all the conceivable mechanisms by which the inhibitor could 

 act to produce the effect observed, in order that the plan will better enable 

 critical experiments to be performed. An ideal plan, perhaps, can be con- 

 structed much like a flowsheet or botanical classification key, in that the 

 results of each test should indicate the nature of the subsequent experi- 

 ment, and so on as the localization progresses. For example, it is usually 

 advisable to begin with a localization to the major pathways of metabolism. 

 If an inhibitor is found to depress bioluminescence, it might act on either 

 the energy-forming systems or the energy-utilizing systems; the first step 

 would be to determine which of these is susceptible. If an inhibitor reduces 

 respiration with glucose as a substrate, it should be determined whether 

 the action is primarily on the glycolytic pathways or on the tricarboxylic 

 acid cycle. The general procedure is to go from the major metabolic systems 

 progressively to smaller and smaller segments until the ultimate site is 

 reached. Of course, if the initial action observed is on a tissue function or 

 in a whole animal, the first step should be a pharmacological analysis of 

 the site of action before the biochemical site is sought. 



