REVERSAL OF ELECTRON TRANSFER IN THE RESPIR.ATORY CHAIN I31 



of quinone under similar experimental conditions are difficult because of 

 the absorbancy change caused by addition of ATP at 275 m^a. However, 

 preliminary studies show quinone to be oxidized and it may be an impor- 

 tant component of the couple involved in DPN reduction. Such changes 

 are consistent with the idea that AIT is entering the respiratory chain in 

 the flavoprotein-pyridine nucleotide region as well as the cytochrome 

 region. Similar etfects ha\ e been demonstrated in the presence of various 

 respiratory inhibitors, for example cyanide, and even in the presence of 

 dithionite. It is found that dithionite does not readilv penetrate the mito- 

 chondrial membrane and thus mitochondrial pyridine nucleotide is not 

 initially reduced, permitting time for studies similar to those indicated in 

 Fig. 14. ^'arious types of mitochondria show this reaction, for instance rat- 

 liver mitochondria and " digitonin " particles have been tested. 



The specificity of the reaction for various nucleotides has been investi- 

 gated and found to be highly specific for ATP ; GTP, UTP, CTP, and ITP 

 show no measurable reduction of pyridine nucleotide or oxidation of 

 cytochrome c under conditions similar to those of Fig. 14. These data 

 support those already indicating that ATP is interacting with the res- 

 piratory chain through the pathway by which oxidative phosphorylation 

 occurs. 



Discussion 



As illustrated by Fig. 14 the interaction of ATP with the cytochromes 

 appears to be rapid, but is much slower than the rate of ferrocvtochrome 

 c oxidation obtained by the rapid flow apparatus. The reaction is, however, 

 quite sensiti\e to inhibitors of the pathway illustrated by Fig. 11 and 

 it has been found that ADP, oligomycin, and phosphate inhibit the oxi- 

 dation of cytochrome c as well as the reduction of DPX. Thus the pathway 

 by which ATP enters the respiratory chain is identified with the pathway 

 of oxidative phosphorylation by its inhibitor sensiti\itv and nucleotide 

 specificity. This pathway, which has been identified with ATPase and 

 ATP-'^-P exchange activities, is acting under these conditions to transfer 

 energy from ATP into oxidation-reduction couples of the respiratory chain 

 — an ATP-electron transferase acti\ity. That the activity of this enzyme 

 system can be measured in the intact mitochondria without acti\ating 

 hydrolysis of one of the intermediates in the sequence of F'ig. 1 1 presents 

 tremendous advantages for two kinds of experiments : (i) to determine the 

 maximal activity of the ATP-electron transferase pathway, and (2) to 

 evaluate the efiFectiveness of reconstituted phosphorylation systems such as 

 those of Polls [17], Pullman, and Lehninger (this svmposium). 



The efficiency with which ATP can convert its energy into electron 

 transfer is of considerable theoretical and practical interest, particularly in 

 connection with theories of active transport and photosynthesis. We have 



