250 A SYMPOSIUM ON RESPIRATORY ENZYMES 



(500-1000 micrograms) prevented the inhibition. But when large 

 amounts of coenzyme were added, the inhibition was prevented 

 only in the system in which it was the limiting factor; that is, the 

 addition of large amounts of diphosphopyridine nucleotide main- 

 tained activity only in the yeast-fermenting system and did not 

 reduce the toxicity of the diamino compounds in the cocarboxylase 

 system; and, conversely, the addition of large amounts of cocar- 

 boxylase maintained activity only in the carboxylase system. These 

 experiments indicate that the inhibition in both systems is a com- 

 petitive one and further suggest that the action of the diamino 

 compounds is on the protein enzyme component rather than on 

 the coenzyme. 



Rat liver cell (slice) and rat hver suspension (brei) oxidations are 

 also inhibited by the p-aromatic diamines. The toxicity gradient 

 of these compounds to surviving liver tissue is similar to that ob- 

 served in the yeast systems. To detect the protection of these rat 

 liver systems by the addition of reducing agents is not practicable, 

 for it was observed that the presence of these diamino compounds 

 in the crude liver enzyme systems catalyzed the oxidation of the re- 

 ducing agents, cysteine and ascorbic acid. Those diamino compounds 

 that are most toxic act as stronger catalysts of the oxidation of the re- 

 ducing agents than do the less toxic compounds in the presence of a 

 liver suspension. It was important to establish the fact that the 

 p-aromatic diamines are not simply non-specific enzyme poisons. 

 The (Z-amino acid oxidase, tyrosinase, cytochrome oxidase, and acid 

 and alkaline phosphatase enzymes were not inhibited by dimethyl- 

 p-phenylenediamine in equivalent concentrations (5 X 10"^ molar). 



In these experiments (see Table 1) the gradations of the toxicity 

 of the diamino split products of methyl derivatives of aminoazo- 

 benzene parallel the carcinogenic potency of the parent molecules. 

 But inasmuch as the list of compounds of this series which have been 

 tested for carcinogenic power is small, further animal experiments 

 with other related compounds are needed to determine whether this 

 apparent correlation is a true one. Furthermore, the evidence that 

 the production of p-aromatic diamino split products from the parent 

 azo molecule in the liver of the rat is concerned in the resulting 

 production of hepatic cancer is entirely indirect. 



Although the mechanism of the inhibition of the protein enz)Tnes 

 in these several systems by the p-aromatic diamine is obscure, it 

 appears that the formation of an oxidation product or products of 

 the reduced compounds is essential to secure this effect. The indirect 



