530 11. LOCALIZATION OF THE SITE OF INHIBITION 



Perhaps the first preparation to test is a mitochondrial susijension from 

 the tissue using pyruvate as the substrate with priming amounts of malate 

 or oxalacetate. If no inhibition occurs within the proper concentration range 

 of the inhibitor, actions on pyruvate oxidase, on the cycle enzymes, or on 

 electron transport are eliminated, and other major systems must be tested. 

 If inhibition is found, the same preparation may be tested with malate or 

 oxalacetate in equimolar concentrations with the pyruvate, so that now 

 the incorporation of pyruvate into the cycle is not dependent on the com- 

 plete operation of the cycle. The degree of inhibition will be indicative of 

 the site in the cycle where a block is produced; e. g., if pyruvate oxidase is 

 the susceptible enzyme, the inhibition will be marked whereas if a-keto- 

 glutarate oxidase is the site of action, the inhibition will be minimal or even 

 absent, at least during the early period of measurement. The inhibitor is 

 then tested with various of the cycle intermediates as substrates. It is 

 usually possible to determine the blocked site in the cycle quite easily and 

 the localization can then proceed to the electron transport system, if an 

 oxidation step is involved, or otherwise to the isolated susceptible enzyme. 

 If the inhibition is upon the oxidation of only one substrate or intermediate, 

 it is evident that those components of the electron transport sequence 

 that are common to all the oxidation systems are not affected. A compari- 

 son of the actions of the inhibitor on succinoxidase and the other oxidation 

 systems is often profitable. If succinate oxidation is preferentially inhibited, 

 this restricts the site of action and may aid considerably in the localization 

 because the succinoxidase can be isolated in a reasonably satisfactory form 

 whereas the other systems must be tested in mitochondria or reconstructed. 

 It is at this point that the use of various electron donors and acceptors, or 

 of difference si^ectroscopy if available, is helpful. 



When a significant inhibition of glycolysis is observed, the subsequent 

 localization may proceed somewhat differently according to the tissue in- 

 volved or the techniques available. The simplest approach is generally to 

 add the various glycolytic intermediates and determine at what point along 

 the sequence the pathway is blocked by the inhibitor. Accumulation 

 of intermediates during inhibition may be tested also or the individual 

 glycolytic enzymes, if they are readily available, can be tested directly. 



The situation that arises when no inhibition can be demonstrated on the 

 major oxidation pathways calls for a consideration of the last two mecha- 

 nisms listed (IV and V). Localization to a block in the membrane reducing 

 the entrance of an important substrate is difficult to establish unless the 

 intracellular concentration of that substrate can be determined. For this 

 purpose, it may be possible to block the metabolism of the substrate with 

 another inhibitor so that significant concentrations within the cell can oc- 

 cur. The last mechanism (V) also presents difficulties for the positive proof 

 of its occurrence. If satisfactory inhibition can be shown on some system 



