532 11. LOCALIZATION OF THE SITE OF INHIBITION 



If there is no marked inhibition of respiration at concentrations decreas- 

 ing growth, an uncoupling of phosphorylations should be tested first be- 

 cause it is relatively easy to do compared to an examination of synthetic 

 and mitotic processes. If uncoui^ling is found not to be the mechanism, it 

 would be advisable to obtain all the cytological information possible on the 

 nature of the inhibited cells in order to note any unique characteristics not 

 observable when the cells are depressed by respiratory inhibitors, anoxia, 

 or lack of substrate. 



Analyses for the total protein or nucleic acid synthesized by the cells 

 are seldom very informative because growth inhibition from whatever 

 cause implies a depression of these basic cell components. Possibly the 

 most helpful technique is the use of labeled compounds to determine the 

 nature of the derangements in synthesis if they occur. The incorporation 

 rates of various amino acids into the cell protein or of nucleotide precursors 

 into nucleic acids can be easily determined, and by chromatographic sep- 

 aration the accumulation of intermediates may be detected. If the struc- 

 ture of the inhibitor is such that it might be participating as an abnormal 

 analog, the inhibitor itself can often be labeled and its metabolic course 

 within the cell traced. Inasmuch as many steps of the synthetic pathways 

 are unknown at present, localization is seldom easy and, indeed, many of 

 the important growth inhibitors act by mechanisms remaining to be discov- 

 ered, despite a large amount of investigation to elucidate the susceptible 

 sites. 



Correlation between the Site of Inhibition and the Effects on Living Tissue 



When a single site of action has been found, it is now necessary to show 

 that inhibition at this locus is responsible for the initial observations. The 

 types of evidence for establishing such a positive correlation have been 

 discussed (page 471). However, it is rare when an inhibitor is specific in 

 its action and blocks at only one site, and therefore one must expect in 

 the course of the localization to find inhibitions of varying degrees on dif- 

 ferent pathways or enzymes. There may not be just one site or mechanism 

 reponsible for the depression of complex processes such as resi^iration, but 

 several actions contributing to various extents. When one ends up with 

 data showing three sensitive enzymes with 50% inhibition obtained on 

 enzyme A at 0.25 mM inhibitor, on enzyme B at 0.78 mM inhibitor, and 

 on enzyme C at 1.2 mM inhibitor, the question arises as to the relative 

 contributions each of these inhibitions makes to the over-all effect in the 

 complete system. From the in vitro results alone it would be rash to assign 

 contributions at any inhibitor concentration because the enzymes may be 

 either more or less sensitive within the cell. If during the localization the 

 data have not already been obtained, one should investigate in the whole 

 system the effects of the inhibitor on intermediate and product concentra- 



