882 17. PLANNING AND REPORTING INHIBITION STUDIES 



enzymes can often be obtained by variation of the pH, and the characteri- 

 zation of an enzyme group is more important than a simple demonstration 

 of inhibition. 



(9) Consider the possibility of nonspecific effects of inhibitors. In the case 

 of weak inhibitors particularly, where reasonably high concentrations must 

 be used, one must attempt to eliminate such effects as might arise from 

 an increase in the ionic strength. The use of comparable concentrations of 

 otherwise inert salts will often reveal that an observed effect is actually 

 a nonspecific salt effect rather than a direct inhibition. 



SUGGESTIONS PARTICULARLY APPLICABLE 

 TO INHIBITIONS IN CELLULAR PREPARATIONS 



(1) Determine and correct for any endogenous metabolism. The presence of 

 a significant degree of endogenous metabolism may influence inhibitions, 

 whether the endogenous metabolism itself is inhibited or not, especially in 

 those cases in which the effects of the inhibitors on the metabolism of a 

 particular added substrate are determined. The means for correcting for 

 endogenous metabolism are discussed in Chapter 9. 



(2) Consider the penetration of the inhibitor into the cells. The effect of 

 an inhibitor on an intracellular enzyme will depend in part on the ability 

 of the inhibitor to penetrate to the enzyme and on the concentration of the 

 inhibitor within the cell. Rough predictions of penetrability can often be 

 made on the basis of the chemical nature of the inhibitor but it is always 

 better, whenever possible, to obtain experimental evidence on the intra- 

 cellular concentration during the inhibition. In this connection, it is fre- 

 quently of value to vary the pH of the external medium if the inhibitor 

 is ionizable, for in this way a dependence on the penetration may be 

 demonstrated. 



(3) Use as far as possible the optimal inhibitor concentration. Choose that 

 concentration, or range of concentrations, of the inhibitor which will most 

 likely inhibit intracellularly to the degree desired, basing your choice on 

 the in vitro actions of the inhibitor and on whatever information is avail- 

 able on the relationship of intracellular to extracellular concentrations. 

 The greater care that is given to these considerations, the greater will be 

 the chance of a selective and meaningful action. 



(4) Use inhibitors, inhibitor concentrations, and conditions wJiich will 

 most likely provide specific effects. One should first consider all the known 

 and possible effects of the inhibitor and choose those conditions, if such 

 are possible, for maximal selectivity of action, as described in Chapter 16. 

 In addition, all the evidence that it is possible to obtain on the specificity 



