METHODS OF LOCALIZATION 521 



Use of a Second Inhibitor 



When several metabolic reactions are proceeding in a preparation, it is 

 occasionally possible to narrow the systems investigated with specific in- 

 hibitors. Any effect of the inhibitor that is being studied can then more 

 readily be attributed to the "isolated" pathway or enzyme. The simplest 

 situation is a monolinear chain. The oxidation of «-ketoglutarate in mi- 

 tochondria is followed by the oxidation of the succinate formed and to 

 some extent by further oxidations. By the proper use of malonate, at a 

 concentration that effectively inhibited succinate oxidation but did not 

 interfere with a-ketoglutarate oxidation, Slater and Holton (1954) were 

 able to study the properties of the a-ketoglutarate oxidase uncomplicated 

 by these subsequent reactions. The blocking of one pathway in a bilinear 

 system will also allow the action of an inhibitor on the other pathway to 

 be studied. An investigation into possible inhibitors of the 0-methylation 

 of the catechol amines, for example, could be done more quantitatively if 

 the monoamine oxidase were blocked. 



Enzyme inhibitors sometimes produce a stimulation of metabolism or 

 cell function (page 451) and the nature of the reactions involved in this 

 may be probed with other inhibitors. Malonate stimulates the respiration 

 of heart slices following an initial depression; the presence of fluoride almost 

 completely prevents this stimulation (Webb et al., 1949 b) suggesting that 

 the rise in respiration is mediated through glycolysis. Malonate also stim- 

 ulates the contractility of hypodynamic ventricle stri])s and this is ef- 

 fectively counteracted by low concentrations of iodoacetate (Covin and 

 Berman, 1956), again pointing to the role of glycolysis in the stimulation. 

 These results, of course, did not exactly localize the site of action of malo- 

 nate but did present some possibilities for further testing, e.g., the elimi- 

 nation of a rate-limiting step in the conversion of glucose to pyruvate. 



A comparison of the actions of two or more inhibitors can be of use in 

 making a localization more certain. If a new inhibitor has been shown to 

 act on a particular enzyme or pathway, the demonstration that it produces 

 the same effects as an inhibitor known to act specifically at this site sub- 

 stantiates the localization. Let us assume that a substance has been shown 

 to inhibit the phosphoglyceraldehyde dehydrogenase. If one wishes to 

 be certain that this is its site of action in the cell, the inhibitor should be 

 shown to have at least some of the effects of iodoacetate, e.g., an accumu- 

 lation of hexosephosphates, a reduction in anaerobic lactate formation, 

 the development of contracture in muscle, or the abolition of retinal ac- 

 tivity. In case the actions of the inhibitors differ, it must be concluded 

 that either the new inhibitor is not inhibiting the dehydrogenase specifi- 

 cally or that the classic concept of iodoacetate action is incorrect. The 

 greater the correlation between the over-all effects of a group of inhibi- 

 tors and the ability of the various members to inhibit a certain reaction. 



