METHODS OF LOCALIZATION 515 



the case when an alternate pathway becomes more important in the me- 

 tabolism of the intermediate, because the products of this alternate path- 

 way may increase in concentration. But there are many more complicated 

 situations where, in the closely integrated metabolism of the living cell, 

 a system other than the one directly attacked will be modified and the 

 concentrations of its intermediates altered. A block in any process in- 

 volved in the formation of high-energy phosphate bonds is apt to induce 

 an accumulation of various precursors for synthetic reactions. Also the 

 reduction in the formation of some product of a sequence may give rise to 

 the accumulation of another compound with which this product reacts. 

 One can easily imagine other circumstances but it should be obvious that in 

 the cell the inhibition of any one process may in a variety of ways affect 

 other processes and the entire steady-state pattern of metabolism be made 

 abnormal. Finally, if the inhibitor is not specific upon a single enzyme, 

 secondary actions may cause rises in the concentrations of intermediates 

 in pathways that are actually not important with regard to the principal 

 mechanism by which the inhibitor produces its effects. 



Addition of Various Substrates or Intermediates 



A comparison of the actions of an inhibitor in the presence of various 

 substrates may provide information on the site of action. If an inhibitor 

 has been found to act on the tricarboxylic acid cycle, the sensitive enzyme 

 can usually be found by simply testing the inhibitor against all the sub- 

 strates and cycle intermediates added individually. Using a reducible ac- 

 ceptor dye it is possible to determine the relative actions of an inhibitor 

 on different dehydrogenases by testing with each substrate alone. Even 

 in intact cells it is often possible to demonstrate different degrees of inhi- 

 bition when the substrate is changed. 



These differences in inhibition may be illustrated by considering some 

 common multienzyme systems. In a monolinear chain: 



I 

 A^B-H^C-^D^E (11-1) 



if the inhibitor acts on the step B -> C, the formation of E will be depressed 

 when A or B are used as substrates, but not when C or D are used. In other 

 words, addition of C or D will overcome the block. This situation is seen 

 in the differential effects of iodoacetate on glucose and pyruvate metabolism. 

 Lundsgaard (1932) found that iodoacetate-poisoned yeast would oxidize 

 pyruvate more readily than carbohydrate and Quastel and Wheatley (1932) 

 reported the same situation in guinea pig brain. More recently it has been 

 shown by Heald (1953) that electrically-stimulated brain slices were in- 

 hibited more potently by iodoacetate when glucose was the substrate 

 than when lactate was the substrate. The differential effect has been studied 



