THE FUNCTIONAL LINK OF SUCCINIC DEHYDROGENASE 1 99 



after the addition of GTP or ITP as compared with that induced by ATP. 

 Furthermore, only trace amounts (less than 5 x iq-^ m) of phosphoenol- 

 pyruvate, the product of the oxaloacetic carboxylase reaction, could be 

 detected after the addition of ATP. 



(c) No measurable amounts of oxaloacetate could be detected after the 

 arsenate-dicoumarol p reincubation ; the assay was sensitive to concentra- 

 tions of oxaloacetate in the incubation mixture as low as 5 x 10 ^^ m [ii]. 



Although these findings seem to preclude a possible involvement of 

 oxaloacetic acid as directly responsible for the observed inhibition of 

 succinate oxidation, some indication was obtained that the presence of 

 oxaloacetate was necessary during the preincubation in order to obtain 

 the inhibited state. Addition of cysteine sulphinate during the arsenate- 

 dicoumarol preincubation, which could be expected to remove all the 

 oxaloacetate formed from endogenous substrates, resulted in a complete 

 protection of succinate oxidation. 



The substrate level phosphorylation compartment and the 

 energy source for the activation of succinate oxidation 



Evidence has been presented that the a-ketoglutarate-linked substrate- 

 level phosphorylation can give rise to an ATP which is not directly available 

 to the DNP-induced ATPase [12]. From this finding the concept was 

 developed that the ATP originating from the substrate level phosphory- 

 lation is compartmentalized in the mitochondria and that accessory reac- 



TABLE II 



Effect of AMP and of Various Substrates on the DNP-Induced Depletion 

 OF Mitochondrial Endogenous Phosphate [12] 



Each tube contained in a final volume of 3 ml.: 0001 M AMP, o-oooi M 

 DNP, 0-003 '^i glutamatc, 0-003 ^1 /S-hydroxybutyrate, 0-003 -^i oxaloacetate and 

 0-003 ^^ succinate; other experimental conditions as in Table I; o-ooi m MgCl., 

 was also added in P^xpt. 2. Time of incubation 7 min. in Expt. i and 5 min. in 

 Expt. 2. 



Additions Counts /sec. 



Expt. I none 429 



DNP 277(65-0%) 



DNP + succinate 170(39-8%) 



DNP + AMP 114(26-7%) 



Expt. 2 none 326 



DNP 238(73-2%) 



DNP + Glutamate 2i2(65-2"o) 



DNP + /3-hydroxybutyrate 223 (68-6%) 



DNP + oxaloacetate 134 (41 - 1 %) 



DNP + succinate 1 30 (40 - 1 %) 



