ATPase in oxidative phosphorylation 



247 



Finally, in agreement with current concepts of the mechanism of 

 oxidative phosphorylation which predict that the ATPase and ^-P— ATP 

 exchange reactions are involved in a common reaction sequence in oxida- 

 tive phosphorylation, it was found that the purified ATPase was required 

 to reconstitute a dinitrophenol sensitive ^-Pj-ATP exchange. Representa- 

 tive data are presented in Table VII. Aliquots of a given particulate 

 preparation were used to measure either the esterification of P; in the 

 presence of succinate or the incorporation of ^'^Pj into ATP in the absence 

 of added substrate. As may be seen, neither the particles nor the purified 

 ATPase when tested alone catalyzed an appreciable ^'^P— ATP exchange or 

 phosphate esterification. However, the addition of increasing amounts of 



TABLE VI 



Effect of Various Compounds on ATPase and Phosphorylation Activity 



Compound 



P:0 



ATPase 



2,4-dinitrophenol Inhibition 



Pentachlorophenol ,, 



/)-Chloronnercuribenzoate ,, 



Azide „ 



Dicoumarol „ 



Dihydrovitamin K^ diphosphate „ 



Triiodo-L-thyronine ,, 



Amytal — 



Potassium cyanide — • 



Potassium fluoride None 



Warfarin None 



Stimulation 

 Inhibition 



None 

 None 

 None 



None 



the purified ATPase to the particles resulted in parallel increases in both 

 of these activities. 2,4-dinitrophenol abolished both reactions. 



Based on the evidence presented, as well as on other supporting data, 

 we have concluded that the catalytic site or sites responsible for the 

 hydrolysis of ATP and for the coupling activity reside on the same protein. 

 It became necessary, however, to explain the observation that during the 

 course of purification the ATPase was purified to a greater extent than 

 the coupling activity. The apparent greater purification of the ATPase was 

 actually not based on the removal of other protein impurities, but depended 

 to a large extent on an absolute increase in total units. These results indi- 

 cate an activation of a hydrolytic site or removal of an inhibitor rather than 

 a physical separation of the two components. 



We look upon the hydrolysis of ATP by the coupling factor as an 

 aberrant activity which the enzyme has acquired following the disruption 

 of the mitochondria. Bound to the structure of the undamaged mito- 

 chondria, the hydrolytic potentialities of this protein are largely masked, 



