. Biological structure and function; proceedings. Biochemistry; Cytology. 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 succ
. Biological structure and function; proceedings. Biochemistry; Cytology. 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 Reduction of acetoacetate by succinate in rat liver mitochondria (Azzone, Ernster, and Weinbach, unpublished).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
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