38 ALBERT L. LEHNINGER 



any sample of digitonin particles to " rebind " soluble ATP-ADP exchange 

 enzyme; this upper limit in molar terms is approximately equal to the 

 total potential ability of the preparations to catalyze phosphate uptake at 

 a P : O ratio of 3. The specific rebinding to phosphorylating assemblies 

 indicates that the soluble ATP ADP exchange enzyme is a part of the 

 coupling machinery. It also indicates that this exchange enzyme has 

 another functional site which is reactive with an as yet unknown "sub- 

 strate " molecule, presumably a preceding enzyme of the coupling sequence, 

 with which ATP and ADP must come into equilibrium. 



In preliminary experiments it has been found that the soluble ATP- 

 ADP exchange enzyme, when added to digitonin fragments giving 

 suboptimal phosphorylation, will significantly increase the P:0 ratio 

 [9,20]. While the effect requires further investigation, it gives further 

 evidence for participation of this enzyme in the mechanism of oxidative 

 phosphorylation. 



M-factor 



With the availability of highly purified preparations of the ATP-ADP 

 exchange enzyme, efforts were begun to establish the nature of the binding 

 of this enzyme to preceding components of the coupling mechanism, in 

 the hope that the chemical nature of the intermediate reactions catalyzed 

 in the energy-coupling sequence could thus be approached. An important 

 lead into the enzymic aspects of the recombination phenomena was 

 afforded by the finding that extracts of mitochondria contain a soluble 

 heat-labile substance of protein nature (designated as M-factor) which 

 when added to normal digitonin particles greatly increases the sensitivity 

 of the inherent ATP-ADP exchange reaction to dinitrophenol [21]. 



Most preparations of digitonin particles are substantially but not 

 completely inhibited by dinitrophenol [16]. The degree of inhibition, 

 which varies from 20-90*^,' (, among different preparations cannot be in- 

 creased simply by increasing dinitrophenol concentrations above the level 

 of approximately 5 x io~^ m (which produces essentially complete un- 

 coupling of oxidative phosphorylation). This finding suggests that the 

 total ATP-ADP exchange activity of any given sample of digitonin 

 particle consists of two components: a "coupled" component, sensitive 

 to DNP, and a "dissociated" or "uncoupled" ATP-ADP exchange 

 activity, which may be a portion of the coupling machinery but which 

 has been " dislocated " from the DNP-sensitive reaction during preparation 

 of the particles. 



Data in Fig. 4 show that addition of soluble partly purified protein 

 fractions from mitochondrial extracts can greatly increase the fraction of 

 the total ATP-ADP exchange activity which is sensitive to dinitrophenol. 



