156 LARS ERNSTER 



insignificant reduction of acetoacetate, probably because of the unfavour- 

 able equilibrium of the malate + acetoacetate ^=i oxaloacetate + /S-hy- 

 droxybutyrate system. This finding, together with the amytal-sensitivity of 

 the succinate-linked reduction, thus clearly eliminated the possibility that 

 malate originating from succinate rather than the latter itself might 

 constitute the reducing agent for acetoacetate. 



As could be expected from the great positive diflFerence in redox 

 potential between the succinate /fumarate and DPNH/DPN couples (cf. 

 [10]), the succinate-linked reduction of acetoacetate in the present system 

 was strictly dependent on an active oxidative phosphorylation coupled to 



Malate 



-Succ.+ amytal 



No substrate 



Malate 

 + amytal 



Succinate 



10 20 



Minutes 



Fig. 10. Reduction of acetoacetate by succinate in rat liver mitochondria 

 (Azzone, Ernster, and Weinbach, unpublished). Each flask contained: mito- 

 chondria from 150 mg. liver, 20 mM glycylglycine buffer, pH 7*5, 8 mM MgCla, 

 62 mM sucrose, 25 mM KCl, 5 mM P,, 5 mM acetoacetate, and, when indicated, 

 10 mM succinate, 10 mM L-malate, 2 mM amytal, in a final volume of i ml. Incuba- 

 tion at 30'^ Acetoacetate determined according to Walker [41]. 



the terminal oxidation of succinate. Accordingly, as shown in Fig. 11, the 

 acetoacetate reduction was abolished by dinitrophenol. It may be noticed 

 that half-inhibition was reached at a concentration of about 5 x io~^ M, 

 which is considerably below that required for a corresponding depression 

 of the oxidative phosphorylation. This finding is in agreement with the data 

 of Chance and Hollunger [10], from their spectrophotometric studies of 

 the succinate-linked reduction of endogenous DPN. Since dinitrophenol 

 is known to abolish respiratory control at a concentration lower than that 

 needed for an actual depression of the phosphorylating capacity [42, 43], 

 these data indicated that the reduction of acetoacetate by succinate in the 

 present system was dependent not only on an active oxidative phos- 

 phorylation but also on a state of respiratory control, the latter allowing 



