MITOCHONDRIA, CELLS, AND TISSUES 



571 



either the enzyme or the substrate are unstable under the experimental 

 conditions. The preincubation time and the conditions must be identical 

 in each of the procedures. When the locus of inhibition is established, a 

 time course for the inhibition should be determined by using different prein- 

 cubation intervals. If K^ or other equilibrium characteristics are to be ob- 

 tained, it is essential to know when the reaction of the inhibitor is complete 

 and equilibrium has been achieved. 



If an activator or coenzyme is also a component of the enzyme system, 

 the procedure is necessarily more comjilex and in addition to the four 

 tests described above the procedures given in the tabulation below must 

 be carried out for a complete analysis: 



The first test (e) is designed to determine if the inhibitor reacts with the 

 activator or coenzyme; (/), {g), and (A) will show if the inhibitor forms a 

 complex with two comi^onents of the system or reacts with the substrate or 

 activator only in the presence of the enzyme; and (^), (j), and {k) are the 

 controls for the three preceding tests. It is suggested that such a series 

 of tests be undertaken before the more quantitative aspects of the inhibi- 

 tion are investigated because the latter can be done efficiently only after 

 the basic mechanism of the inhibition is established. 



RATES OF INHIBITION IN MITOCHONDRIA, CELLS, AND 



TISSUES 



Interpretation of the rate at which an inhibition develops may be very 

 difficult when the enzyme that is attacked by the inhibitor is located within 

 a cell and is separated from the ambient medium by one or more proto- 

 plasmic membranes. Actually very little really quantitative work on the 

 kinetics of intracellular inhibition has been done. However, the rates of 

 drug action have been the subject of much study and speculation, and the 

 results and conclusions from this branch of pharmacodynamics are fre- 

 quently valuable for investigations of metabolic inhibition since funda- 

 mentally the same problems are involved. The best discussion of the rates 

 of drug action remains that of Clark (1937) and there he summarizes his 



