SPECIFICITY OF INHIBITION 873 



differences in pH and the acidic or basic nature of the inhibitor. In other 

 cases it may be worthwhile to analyze for the inhibitor within the cells. 

 With inhibitors that penetrate through the f)lasma membrane slowly, it is 

 necessary to i^rovide enough time for equilibrium to occur. 



{D) The lowest concentrations of the inhibitor to produce significant 

 effects should be used rather than concentrations aimed at nearly complete 

 inhibition. It is also of value to construct a concentration-action curve 

 for the cellular inhibition and not use only a single concentration of the 

 inhibitor, particularly if correlations between enzyme inhibition and func- 

 tional disturbance are sought. 



One can seldom be absolutely certain of specificity in cellular studies 

 but every effort should be made to approach this goal. 



Use of Two or More Inhibitors to Improve the Specificity 



There are two general ways by which the judicious use of additional in- 

 hibitors may increase the siDecificity of the primary inhibitor. The first 

 way is limited to certain types of systems. It is to use a second inhibitor 

 for the purpose of blocking an interfering reaction. For example, in the 

 study of the inhibition of a-ketoglutarate oxidation, malonate may be add- 

 ed to block the further oxidation of the succinate formed from the a-keto- 

 glutarate, and in this way the first reaction can be in a way isolated and any 

 effects of the inhibitor being tested must be on the a-ketoglutarate oxidase. 

 In the same way, the inhibition of certain anaerobic processes can be in- 

 vestigated during a block of the oxidative reactions with cyanide. By this 

 technique the inhibitor is not made more specific but the range of systems 

 on which it can act is reduced. 



The other use of two or more inhibitors is more general and theoretically 

 could be used to improve any specificity. This has already been men- 

 tioned in Chapter 10 where the actions of multiple inhibitors were treated. 

 If two inhibitors act on one enzyme but their further inhibitions are on 

 different enzymes, the use of them together will increase the specificity 

 with respect to any of these other enzymes. Put simply, if I^ acts on E^ 

 and E2 whereas I2 acts on E^ and Eg, I^ + I2 will inhibit E^ more potently 

 without inhibiting E2 or Eg more than with the single inhibitors. This is 

 the principle on which drug combinations are used in medicine with the pur- 

 pose of increasing the beneficial actions while decreasing the side effects. 

 We shall now investigate the conditions under which such a combination of 

 inhibitors can give significant improvement in the specificity and determine 

 the proper concentrations of each inhibitor to use. 



The various inhibitions for a system of two enzymes and two inhibitors 

 of the type described above may be written as follows. 



