ATPase in oxidative phosphorylation 243 



only if the system was supplemented with both DPX and glutamic de- 

 hydrogenase. Again, both /S-hydroxybutyrate and glutamate were oxidized 

 without uptake of P; unless the coupling factor was present. The maximal 

 P:0 ratio obtained was independent of the nature of the substrate under- 

 going oxidation, suggesting that only phosphorylation sites in the respira- 

 tory chain between succinate and oxygen contribute to the P:0 ratio. A 

 more precise localization of the phosphorylation site(s) is currently under 

 investigation. 



Purification and characterization of the coupling factor revealed the 

 presence of a Mg^ ^-dependent, dinitrophenol-stimulated ATPase. The 

 generallv accepted concept that the mitochondrial ATPase is functionally 



TABLE II 



Effect of the Purified Coupling Factor on Phosphorylation Associ.\ted 

 WITH THE Oxidation of Various Substrates 



The experimental conditions have been previously described (cf. Table II [4]). 



o 1 Coupling ^ , r, ^ r. ^ 



Substrate . O., uptake R uptake P :0 



factor ■ ^ ' ^ 



fiA /min . /mg. /u A /min . mg. 



Succinate 

 DL-isocitrate 

 DL-^-hydroxybutyrate 

 L-glutamate 



related to the enzymic mechanism of oxidati\e phosphorylation prompted 

 us to examine the relationship between the ATPase and coupling activities. 

 Since the most highly purified preparations which hvdrolvzed 80 to 100 

 /^moles of ATP min. mg. protein induced phosphorylation coupled to 

 oxidation in the presence of the particles, the question arose whether these 

 activities were located in the same protein. 



ATPase activity, which was measured in a system using phosphoenol- 

 pyruvate and pyruvate kinase as a regenerating system for ATP, was 

 markedly higher in the presence than in the absence of the regenerating 

 system. This is due, at least in part, to the fact that the pyruvate kinase 

 system removes the ADP which is inhibitory to the enzyme. ATPase 

 activity was increased 50 to 75",, by the addition of 5 x 10 -* m 2,4- 

 dinitrophenol. 



Studies on the purified ATPase revealed properties consistent with its 

 participation in coupled phosphorylation and similar to those described 



