FUN'CTION OF FLAVOENZYMES IN ELECTRON TRANSPORT l6l 



respiratory increase and the acetoacetate reduction) the corresponding 

 values were 2 • 40 /xatoms and 5 • 7 /xmoles, respectively. Addition of ATP, 

 hexokinase and glucose, which abolished acetoacetate reduction, also 

 significantly diminished the respiratory stimulation due to acetoacetate, 

 to the value of o • 8 ^uatom ; and, finally, addition of oligomycin A to the 

 hexokinase-glucose system restored both respiratory stimulation and 

 acetoacetate reduction to about their original levels, 2-96 /xatoms and 5-4 

 /xmoles, respectively. Thus, in all three cases where acetoacetate reduction 

 occurred, there occurred a respiratory stimulation as well, the rate of 

 which was approximately o • 5 /xatom oxygen per /xmole acetoacetate 

 reduced. Assuming a P/0 ratio of 2 for the aerobic oxidation of succinate 

 to fumarate, this implies a ratio of one high energy bond equivalent per 



TABLE XI 



Effect of Respiratory Chain Inhibitors on Succinate-linked 

 Reduction of Acetoacetate in Rat Liver Mitochondria 



(Azzone, Ernster, and Weinbach, unpublished) 



In each flask: 5 niM acetoacetate, 25 mM succinate, 62 m.M sucrose, 50 niM KCl, 

 20 mM tris buflfer, pH 7 5, 8 mM MgCL, mitochondria from 400 mg. liver. 

 When indicated : 5 m.M .ATP, 2 mM amytal, i ■ 25 /tg./ml. antimycin A, 1-25 /xg./ml. 

 oligomycin A, 0-5 mM KCN. P'inal \olume, 2 ml. Cjas phase, air. Temperature, 

 30". Time of incubation, 20 min. 



.Additions 



J.-\cAc (/xmoles) 



ATP +ATP 



None 3-4 3-2 



Antimycin A 00 



Cyanide o o 



No as gas phase o o 



Oligomycin .A 3-9 40 



molecule of acetoacetate reduced. This value is in agreement with that 

 envisaged by the reaction mechanism for the succinate-linked reduction 

 of DPN, discussed above (cf. Reactions i and 2, and Fig. 9), and is 

 considerably lower than those previously arrived at by Chance and 

 Hollunger [10] and by Chance [47]. 



As shown in Table XI, the succinate-linked reduction of acetoacetate 

 in the present system was completely abolished by the respiratory inhibi- 

 tors, antimycin A and cyanide, as well as in anaerobiosis. Added ATP, 

 which had no effect on the aerobic system, did not remove these inhibi- 

 tions. Hence, in contrast to the succinate-linked reduction of DPN in the 

 high-energy phosphate-depleted mitochondria (cf. Fig. 10), the succinate- 

 linked reduction of acetoacetate in non-depleted mitochondria appears to 



VOL. II. M 



