36 ALBERT L. LEHNINGER 



its soluble form to the mechanism of respiratory energy coupling. In intact 

 mitochondria [18] or membrane fragments [16] the ATP-ADP exchange 

 is sensitive to DNP, but only indirectly, as pointed out above. If the 

 soluble ATP-ADP exchange enzyme could be "reconnected" with the 

 DNP-sensitive reaction, the soluble enzyme might regain its DNP- 

 sensitivity. We have now found it possible to reconfer DNP-sensitivity on 

 the soluble form of the ATP-ADP exchange enzyme quite simply by 

 adding it to fresh preparations of digitonin fragments [19]. The typical 

 experiment illustrated in Fig. i shows that addition of the DNP-insensitive, 

 soluble exchange enzyme to fresh rat liver digitonin fragments in which the 



500 



E 

 < 



200 



i 



DP SOL COMBINED EXPECTED 



ENZ FOUND IF NO 



INTERACTION 



Fig. I. "Recombination" of soluble, DNP-insensitive ATP-ADP exchange 

 enzyme with digitonin particles to restore DNP-sensitivity. System contained 

 o-oi M ATP, o-oo6 M ["C]-ADP, and 5 x 10-^ m DNPwhere shown. Black portion 

 of bars indicates fraction of activity sensitive to DNP. 



inherent nucleotide exchange activity is inhibited significantly by dinitro- 

 phenol, causes, in the combined system, a summation of the total exchange 

 activities in the absence of DNP. However, it is evident that a very large 

 fraction of the combined ATP-ADP exchange activity is now sensitive to 

 dinitrophenol, to a far greater extent than would be expected by simple 

 addition of the two reactions measured separately. Many experiments of 

 this kind thus demonstrate the conferral of DNP sensitivity on the soluble 

 nucleotide exchange reaction by adding it to fresh digitonin particles. 

 Dinitrophenol sensitivity is not conferred on the soluble enzyme by aged 

 digitonin particles, which are incapable of oxidative phosphorylation 

 (Fig. 2). Furthermore the DNP-sensitivity of the recombined system is 

 abolished in the presence of azide, which, as mentioned above, can dis- 

 sociate the particulate ATP-ADP exchange from the DNP-sensitive site 

 (Fig. 3). The conferral of DNP-sensitivity on the soluble ATP-ADP 

 exchange enzyme is thus specific and this finding establishes the identity 



