126 BRITTON CHANCE 



with the diminution of respiratory rate following phosphorylation of ADP. 

 In Fig. ID, however, the amytal-treated material shows pyridine-nucleo- 

 tide oxidation that does not reach a steady state for approximately i min. 

 In fact, most of the ADP has been phosphorylated before DPNH oxi- 

 dation is completed. Subsequently, upon exhaustion of ADP, pyridine- 

 nucleotide reduction proceeds for approximately i min. While it would be 

 expected that oxidation of succinate-linked DPNH would be sensitive to 

 amytal, in view of the general sensitivity of DPNH-linked oxidations to 

 this inhibitor, it is surprising that the reduction is so severely inhibited 

 unless the reaction is proceeding by reversed electron transfer through 

 the same carrier as that through which the oxidation reaction is occurring. 



340-374m/x 



ADP 



log Iq/I = 0005 



State 4' / 007/xM 



(succinote > — / PN/sec 



+ 0-8mM amytal) 



50sec 



Fig. 10. Effect of amytal upon cycle of oxidation and reduction of succinate- 

 linked reduced pyridine nucleotide in presence of o • 8 mM amytal. Experimental 

 conditions identical to those in Fi^. 3 and comparison of rates of reaction in both 

 cases is possible (Expt. 683-3). (Reproduced with permission of The Journal of 

 Biological Chemistry.) 



We interpret this experiment as identifying an amytal-sensitive reaction 

 in not only the oxidation but also the reduction of pyridine nucleotide. 

 This result strongly supports the idea that energy-linked reversal of 

 electron transfer through the respiratory carriers is involved in the 

 succinate-linked reductions of DPN (Figs. 5, 8). 



The question of the level to which electron transfer proceeds toward 

 oxygen before it is bypassed into pyridine-nucleotide reduction is sugges- 

 ted by experiments in which reduction of DPN by succinate and ATP is 

 highly inhibited by hydroxyquinoline-N-oxide (HOQNO) or antymycin- 

 A. At the present time experimental data suggest that electron transfer 

 proceeds to the antimycin-A-sensitive point (Fig. 8). 



