198 GIOVANNI FELICE AZZONE 



mitochondrial high energy phosphate was almost completely abolished in 

 the presence of 2 mM amytal. In agreement with this experiment, it was 

 found (Fig. 4) that when amytal was added prior to, or together with, 

 arsenate a high respiration ensued upon the addition of succinate. On the 

 other hand, if amytal was added afler the preincubation with arsenate, the 

 succinoxidase activity was greatly inhibited and again, added ATP was 

 required to increase the oxidation rate. 



It could be demonstrated clearly that the depression of the capacity for 

 succinate oxidation was caused by the depletion of mitochondrial high 

 energy phosphate compounds, and not by the presence of arsenate itself. 

 This was accomplished by the use of 2,4-dinitrophenol (DNP) plus AMP 

 to pretreat mitochondria which were then washed free of the depleting 

 agents. The succinoxidase activity of these preparations was very low and 

 could be stimulated more than two-fold by the addition of ATP (Fig. 5 {a)). 

 Inorganic phosphate also could increase the oxidation rate but only if 

 added before the uncoupling agent and together with succinate (Fig. 5 {b)). 

 Under these conditions high energy phosphate compounds could be 

 synthetized by the mitochondria. 



The question of oxaloacetate 



Oxaloacetate is known to be a competitive inhibitor of succinic de- 

 hydrogenase, and its accumulation has been considered as chiefly 

 responsible for the inhibitions of succinate oxidation observed by difi^erent 

 workers. Furthermore, a protective effect of ATP against the inhibition 

 induced by oxaloacetate has been reported by Pardee and Potter [8] and 

 by Tyler [9]. Therefore it was necessary to examine in greater detail the 

 mechanism by which energy is provided for the activation of succinate 

 oxidation, and also the possible ways by which oxaloacetate may interfere 

 with the mitochondrial oxidation of succinate. 



The possibility that the accumulation of oxaloacetate, per se, could be 

 responsible for the low rate of succinate oxidation after the arsenate- 

 dicoumarol preincubation, has been excluded by three types of experiments : 



{a) The inhibition was not relieved by the addition of cysteine 

 sulphinate, in the presence of amytal. Control experiments showed that 

 cysteine sulphinate did remove an inhibition of succinate oxidation due to 

 added oxaloacetate in agreement with the finding of Singer and Kearney 

 [10] who have demonstrated that cysteine sulphinate transaminates 

 oxaloacetate to aspartate. 



{b) It would be anticipated that ATP could be replaced by GTP (or 

 ITP) if ATP was acting by removing oxaloacetate via the oxaloacetic 

 carboxylase reaction since this reaction specifically utilizes GTP (or ITP). 

 Under our conditions little stimulation of succinate oxidation was observed 



