472 9. INHIBITION IN CELLS AND TISSUES 



of a connnection of function with these metabolic pathways. It is, however, 

 often very difficult to establish a direct correlation between the metabolic 

 and functional changes for reasons given above; in addition the lack of 

 absolute specificity for most inhibitors complicates the matter still further. 

 Despite the serious problems in such investigation, there are several ways 

 in which a correlation may be made with varying degrees of certainty. 



I. Correspondence of inhibitor concentrations used. The concentrations 

 of inhibitor necessary to produce the enzymic and functional changes must 

 be comparable. The use of concentrations far in excess of that inhibiting 

 on isolated preparations, as is often done, eliminates the possibility of 

 correlation, unless evidence can be produced that the intracellular concen- 

 tration is lower than the external concentration and similar to that effec- 

 tive in vitro. 



II. Demonstration of specificity at the appropriate concentration. When 

 the inhibitor at the concentration causing functional change can be shown 

 to be specific in action on one enzyme or metabolic pathway, and in all 

 probability without nonenzymic action, the relationship between this 

 function and the enzyme attacked is more certain. However, there are very 

 few instances where reasonable specificity can be attributed to an inhibitor. 



III. Simultaneous demonstration of functional and metabolic change. Oc- 

 casionally it is possible to show that during the change in function there 

 is a concomitant inhibition of a particular enzyme or metabolic process, 

 as by the demonstration of the accumulation of some intermediate analyt- 

 ically or spectroscopically, or the failure in formation of some product. 

 Of course, this shows only that the two effects are occurring simultaneously 

 and does not prove that a relation exists betweem them. 



IV. Reversal of functional change by reversal of inhibition. One of the 

 best ways, where it is possible, to make correlation is to show that an 

 alleviation of the inhibition is accompanied by restoration of normal func- 

 tion. If the inhibitor is blocking a pathway, the addition of an intermediate 

 past this block may reverse the functional change. The addition of pyru- 

 vate during glycolytic inhibition by iodoacetate is commonly used to show 

 that the inhibition is indeed on glycolysis. It is necessary to perform the 

 appropriate controls, particularly the effect of addition of the intermediate 

 without the inhibitor, to establish that the effect is reversal of the block. 

 The reversal of inhibition and functional alteration by adding a substance 

 that combines with and removes the inhibitor reveals nothing. 



V. Isolation of the inhibited enzyme. When the enzyme inhibition is reason- 

 ably irreversible, it is sometimes ijossible to extract the enzyme in question 

 from the tissue whose function has been altered, and demonstrate a degree 

 of inhibition on it. This merely shows that both events occur and does not 

 establish caiisal relationship. 



