FUNCTION OF FLAVOENZYMES IN ELECTRON TRANSPORT 1 55 



ENDERGONIC REDUCTION OF ACETOACETATE BY 

 SUCCINATE IN LIVER MITOCHONDRIA* 



Up to the present, the succinate-hnked reduction of mitochondrial 

 DPN has exclusively been studied by measuring the increase in the steady- 

 state level of endogenous DPNH, ensuing upon the addition of succinate 

 and or ATP. One obvious limitation of this system, emerging from the 

 above reasoning, may be that any increase in the DPNH level that one 

 observes is a resultant of two, independent, reaction capacities; on one 

 hand, the capacity of the reaction(s) feeding in electrons from succinate 

 into the respiratory chain (Reaction i in the above formulae), and on the 

 other, the capacity of the chain of reactions by which these electrons are 

 transferred from their point of entrance to oxygen (Reactions 3 and 4). 

 Clearly, if the latter capacity is equal to or exceeds the former, no increase 

 in the level of DPXH may be expected to occur. This situation may 

 render the reproducibility of the observations, e.g. from one tissue or one 

 set of conditions to another, dependent on irrelevant circumstances, and in 

 particular, it may render unrealistic a quantitative evaluation of the 

 number of high-energy bond equivalents required for the reduction of one 

 molecule DPX bv succinate. Moreover, using the above test system, no 

 conclusive evidence has yet been presented that the reducing equivalents 

 appearing in DPXH actuallv originate from succinate and not from some 

 endogenous substrate, the oxidation of which has been facilitated, in a 

 secondary manner, by succinate and /or ATP. 



To overcome these difficulties, it was felt desirable to devise a system 

 in which the DPX reduced by succinate was continuously reoxidized by 

 suitable means, e.g. bv wav of the reversal of a DPX'^-linked dehydrogenase 

 reaction. The reversal of the /S-hydroxybutyric dehydrogenase reaction, 

 consisting of a reduction of acetoacetate to /3-hydroxybutyrate, was 

 considered to be con^•enient for this purpose, since this is the only known 

 reaction by which free acetoacetate can be metabolized in rat liver mito- 

 chondria. It was in fact found that when isolated rat liver mitochondria 

 were incubated in the presence of succinate and acetoacetate under aerobic 

 conditions and in the absence of phosphate acceptor, there occurred a 

 substantial disappearance of acetoacetate which was linear with time and 

 strictly dependent on the presence of succinate (Fig. 10). Furthermore, 

 the acetoacetate reduction was completely inhibited by 2 niM amytal, 

 indicating that it involved an electron transfer between the site of entrance 

 of electrons from succinate into the respiratory chain and DPX. Replace- 

 ment of succinate bv malate, with or without amvtal, resulted onlv in an 



* The studies reported in this and the following section have been conducted 

 in collaboration with Drs. G. F. Azzone and E. C. Weinbach. 



