74 J. B. CHAPPELL 



Either 5 mM-malonate or ^-chlorovinylarsenious oxide (0-5 /xg./ml.) 

 severely inhibited isocitrate oxidation even in the low phosphate medium. 

 These inhibitory effects were largely reversed by the addition of low 

 concentrations of the dicarboxylic acids mentioned above. Half maximal 

 effect was obtained with 2 x io~* M-malate. With mitochondria which 

 had been depleted of their endogenous substrates, or in the presence of 

 malonate or ^-chlorovinylarsenious oxide, neither acetoacetate nor 

 pyruvate together with bicarbonate were able to restore isocitrate oxidation. 



These results suggest that the pyridine nucleotide reduced by the 

 isocitrate dehydrogenase is reoxidized by coupling with the malate 

 dehydrogenase. Neither the ^-hydroxybutyrate dehydrogenase nor the 

 malic enzyme appears to be able to do this. The scheme outlined in Fig. 4 

 is thought to represent the sequence of events occurring in the oxidation 

 of isocitrate by liver mitochondria. This scheme, besides accounting for 



tate-*^ ^^-Reduced— ^ /-^^Oi 



: ^^j^Oxidized^^j^HjO 



I 



TCA cycle 



(1) /socitrate dehydrogenase (3) Malate dehydrogenase 



(2) Pyridine nucleotide transhydrogenase (4) Cytochrome system 



Fig. 4. The proposed pathway of isocitrate oxidation. 



the experimental observations which have been given above, also takes 

 into account the following facts: (i) there exists in liver mitochondria a 

 triphosphopyridine nucleotide (TPN)-linked and not a diphosphopyridine 

 nucleotide (DPN)-linked dehydrogenase [3, 4], (2) the DPN specificity of 

 the mitochondrial malate dehydrogenase [5]. 



Similar results to those given above have been obtained with kidney 

 mitochondria. 



COMPARISON OF GLUTAMATE AND ISOCITRATE OXIDATION 



In liver mitochondria two possible routes of glutamate oxidation are 

 available, one involving glutamate-aspartate transaminase, the other 

 utilizing glutamate dehydrogenase [6, 7]. The transaminase pathway is 

 analogous to the pathway which appears to exist for isocitrate oxidation; 

 both are coupled oxido-reductions and both involve the utilization cf 

 oxaloacetate. The former uses pyridoxal phosphate as a cofactor, the 

 latter pyridine nucleotide. The pathway for glutamate oxidation involving 

 the use of the dehydrogenase, leads to the reduction of pyridine nucleotide, 



