l6o LARS ERNSTER 



where it is utilized for DPN-reduction is apparently not affected by the 

 oligomycin A-block. Accepting the above mode of action of oligomycin A, 

 this would mean, either that this transfer can take place directly, without 

 the intermediary of ATP, or that it proceeds via a fraction of intramito- 

 chondrial ATP which is not available to hexokinase and glucose and whose 

 interaction with the primary high energy intermediates is not blocked by 

 oligomycin A. 



Based on the conclusion, reached above, that "loosely-coupled" 

 respiration could not contribute energetically to the succinate-linked 

 DPN-reduction, it was considered possible to estimate the stoicheiometry 

 of the DPN-reducing system by measuring the difference in rate of 

 succinate oxidation, observed in the presence and absence of added 

 acetoacetate. It may be assumed that the respiration observed in the 



TABLE X 



Stimulation of Succinate Oxidation due to Reduction of 

 Acetoacetate in Mitochondria in Controlled State 



(Azzone, Ernster, and Weinbach, unpublished) 



Experimental conditions as in Table VIII, except that mitochondria from 

 400 mg. liver and 25 mM P, were added per flask. All flasks contained 8 mM MgCl2. 

 Oligomycin A, when present, was added in a concentration of i /xg./ml. 



Additions 



O2 consumption, /xatoms /xmoles AcAc 



With Without 

 AcAc AcAc 



JO, Reduced 



None 9 '78 7-68 2-10 4-7 



Oligomycin A 9-11 6-71 2-40 5-7 



ATP, hexokinase, glucose i9"3 18-5 o-8 o 

 Oligomycin A, ATP, 



hexokinase, glucose iO'i3 7'i7 2-96 5-4 



absence of both phosphate acceptor and acetoacetate, being "loosely- 

 coupled", cannot contribute energy to the reduction of DPN; and conse- 

 quently, that addition of acetoacetate to the phosphate acceptor-free 

 system would result in an increase of the respiratory rate, in a phosphate 

 acceptor-like manner, to the extent it "trapped" energy from the respira- 

 tory chain by reoxidizing endergonically reduced DPN. Data presented in 

 Table X are the mean values of duplicate runs and are reasonably precise. 

 It is seen that addition of acetoacetate to the phosphate acceptor-free 

 system resulted in an increase in oxygen consumption by 2-10 /xatoms, 

 and this was accompanied by a disappearance 4-7 /xmoles of acetoacetate. 

 In the presence of oligomycin A (which slightly stimulated both the 



