96 1- lODOACETATE AND lODOACETAMIDE 



tion completely in yeast, but when acetate is added the inhibition is much 

 reduced (Schmid, 1958). This was taken as evidence that iodoacetate inhib- 

 its glycolysis selectively. Essentially the same was seen in Allomyces mac- 

 rogynus by Bonner and Machlis (1957), 1 mM iodoacetate inhibiting glu- 

 cose respiration 90% and acetate respiration 29%, while 0.1-0.5 mM inhib- 

 its glucose respiration without significant effect on acetate oxidation, and 

 this was again used as evidence for a specific block of 3-PGDH, acetate 

 bypassing this block. However, it must be realized that acetate also by- 

 passes pyruvate oxidase, and acetate kinase is apparently quite resistant 

 (Table 1-13), so that the results with acetate cannot be extrapolated to the 

 normal situation in which pyruvate is the major substrate for the cycle. 



The inhibition of pyruvate oxidation could be due to inactivation of co- 

 enzyme A or lipoate. Shimi and Nour El Dein (1962), for example, postulat- 

 ed that the accumulation of certain cycle intermediates in Aspergillus ter- 

 reus treated with iodoacetate at 0.01 mM might be due to reaction with 

 coenzyme A. The initial work of Lynen et al. (1951) has often been taken 

 as indicating that coenzyme A is readily inactivated by iodoacetate, but the 

 conditions of the experiment have usually been overlooked. The reduced 

 coenzyme A was incubated with 33 mM iodoacetate at pH 7 and room 

 temperature for 2 hr; less than 5% of the coenzyme A activity remained 

 as tested in an acetylation reaction. Whether significant reaction would take 

 place with iodoacetate at 1 mM or below is not known, and experiments 

 along this line with both coenzyme A and lipoate are needed. It requires 

 17 mM iodoacetate to reduce the coenzyme A activity in yeast by 25%, 

 and 43 mM to reduce it 50% (Estler et al, 1960). It has been shown by 

 Davenport et al. (1956) that mouse stomach incubated with iodoacetamide 

 exhibits no reduction of coenzyme A activity over 10 min until a concen- 

 tration of 8 mM is reached (— 30%), and that no inactivation of lipoate 

 occurs with 5 mM iodoacetamide over 30 min. These results indicate that 

 within tissues the inhibitions brought about by iodoacetate and iodoacet- 

 amide are not related to carboxymethylation of these cof actors, and that 

 the inhibition of pyruvate oxidation is probably through reaction with SH 

 groups on the apodehydrogenase. 



Summarizing the situation with respect to the relative inhibitions of the 

 EM pathway and pyruvate oxidation, one can safely state that it is prob- 

 ably impossible to obtain a completely selective action on the EM path- 

 way (i.e., 100% depression of pyruvate formation without an effect on 

 pyruvate oxidation), and that in general it is very difficult to achieve mark- 

 ed inhibition of the EM pathway without to some extent suppressing pyru- 

 vate oxidation. The problem is complicated by the variable sensitivities of 

 the two systems in different organisms and tissues; in some cells it is likely 

 that much greater specificity can be obtained than in others. Another im- 

 portant factor is time. The EM pathway seems to be blocked more rapidly 



