CHAPTER 11 



LOCALIZATION OF THE SITE OF INHIBITION 



It is commonly found that a substance interferes with some phase of 

 metabolism or produces on cells, tissues, or animals effects whose origin 

 is believed to be biochemical. The basic problem of finding the site or sites 

 of the action then arises. One would prefer to localize the action to a single 

 enzyme or metabolic event and on this basis be able to explain the meta- 

 bolic disturbance or the functional changes observed in the presence of the 

 substance. The purpose of this chapter is to present some of the principles 

 and techniques by which such analyses may be carried out and to illustrate 

 these methods with a few examples of localization. No one should imagine 

 that this is an easy problem. There are many often-used inhibitors whose 

 exact site of action is not yet known; for example, phlorizin, antimycin, 

 quinones and naphthoquinones, cadmium and lead, diethyldithiocarbamate, 

 maleate, dinitrophenols, urethane, and alloxan, to name only a few. The 

 action of a new inhibitor may very well not be on a known metabolic sys- 

 tem. When fluoride and iodoacetate were first studied on muscle metabo- 

 lism, the glycolytic pathway had not been elucidated; indeed, these inhi- 

 bitors played a role in establishing the enzyme sequence in glycolysis. 

 Thus the investigation of a new inhibitor will occasionally bring to light 

 a new pathw^ay or enzyme. Although such a contribution to biochemical 

 knowledge may be important, it makes the eventual determination of the 

 site of action particularly difficult. It is really only under fortuitous cir- 

 cumstances that a satisfactory localization can be readily made. 



METHODS OF LOCALIZATION 



Certain procedures are often useful for localization. It is necessary to 

 inquire into the applicability and validity of these methods, which are 

 based to a large extent on the principles discussed in previous chapters. 



Accumulation of Intermediates 



When an inhibitor exerts a blocking action on an enzyme in a metabolic 

 pathway, it would be m'edicted that the intermediate acted upon by this 



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