164 LARS ERNSTER 



Since Reaction 6, but not Reaction 5, is inhibited by amytal, it could be 

 expected that, if Reactions 6 and 7 were occurring, the disappearance of 

 a-ketoglutarate would be diminished in the presence of amytal. The data 

 in Table 14 show that, indeed, there occurred an amytal-sensitive a-keto- 

 glutarate utilization; however, unexpectedly, this was dependent on the 

 presence of externally added ATP. As anticipated, on the other hand, 

 the amytal-sensitive part of the a-ketoglutarate utilization could be 

 completely abolished by hexokinase and glucose. Further data, not included 

 in Table XIV, show that the CO., production, in contrast to the a-keto- 

 glutarate utilization, was not influenced by amytal. All these data are thus 

 consistent w^ith the above reaction sequence. 



TABLE XIV 



ATP-DEPENDENT AmINATIVE REDUCTION OF a-KETOGLUTARATE BY 



Succinate in Anaerobic Mitochondria 

 (Azzone, Ernster, and Weinbach, unpublished) 



In each flask: 0-05 M KCl, 0-02 M glycylglycine buffer, pH 7-5, 8 mM MgClg, 

 10 mM Pj, 5 mM NH4CI, 5 mM a-ketoglutarate, mitochondria from 150 mg. liver. 

 When indicated : 2 mM amytal, 5 or i mM ATP (without or with hexokinase- 

 glucose). Final volume, 2 ml. Temperature 30^. Time of incubation, 20 min. Gas 

 phase. No 



However, regarding the requirement for external ATP, it is obvious 

 that, from the point of view of energy-transfer, this system is funda- 

 mentally different from the succinate-linked reduction of acetoacetate. The 

 role of ATP cannot be that of merely "tightening" the mitochondrial 

 structure (as in the case of the Mg+ +-deficient acetoacetate system, cf. 

 Table IX), since, in the present case, oligomycin A counteracted the ATP 

 effect. It was also found in preliminary experiments that ATP could be 

 replaced by catalytic amounts of AMP. This eliminates the possibility that 

 the a-ketoglutarate-linked phosphorylation might not be able to furnish 

 energy to the succinate-linked reduction of DPN. Whether this discrepancy 

 in ATP requirement between the acetoacetate and a-ketoglutarate systems 

 is due to the different dehydrogenases, /S-hydroxybutyric and glutamic, or 

 to the different sites of phosphorylation, respiratory chain and substrate 

 level, involved in the two systems, cannot be decided at this time. In any 



