248 MAYNARD E. PULLMAN, HARVEY S. PENEFSKY AND E. RACKER 



and during oxidative phosphorylation the enzyme functions primarily as a 

 transfer agent. A similar suggestion was made many years ago to explain 

 the latent ATPase of intact mitochondria [8-10]. 



We feel that the most logical site for the action of this enzyme in 

 oxidative phosphorylation would be the terminal transphosphorylation 

 step. The possibility, therefore, of an ADP-ATP exchange catalyzed by 

 this enzyme was explored. Numerous attempts under various experi- 

 mental conditions have thus far been unsuccessful. However, these 



TABLE VII 



Effect of the Coupling Enzyme on Oxidative Phosphorylation and the 

 ^-Pj-ATP Exchange Reaction 



The particulate fraction was preincubated with the coupling enzyme and Mg + + 

 as described previously [4]. Aliquots of the preincubation mixture containing 

 0-580 mg. of the particulate fraction and the indicated amount of the coupling 

 enzyme were added to the Warburg vessel for the assay of oxidative phosphoryla- 

 tion or to test tubes for the measurement of the ^-Pj-ATP exchange reaction. 

 Oxidative phosphorylation was measured at 30° for 30 min. with succinate as 

 substrate [4]. The ^-Pj-ATP exchange reaction was measured as described else- 

 where [4]. Each tube contained o-oi6 !m ATP, o-oi6 m MgCU, o-oi m tris, pH 

 7-4, 0-04 M ^-Pj (i-2 X 10^ c.p.m.//Lxmole), o-ooi M EDTA, pH 7-4 and the 

 preincubated enzyme mixture in a final volume of o • 5 ml. When added, dinitro- 

 phenol was 5 x 10^ m. 



Coupling 

 enzvme 



O2 

 uptake 



Pi 

 uptake 



P:0 



3-P,-ATP 



failures are not considered decisive in view of the predominance of the 

 hydrolytic activity exhibited by the purified enzyme. Since azide was 

 found to inhibit the ATPase activity at concentrations which do not un- 

 couple phosphorvlation, attempts were made to demonstrate the ^^C- 

 ADP-ATP exchange in the presence of this compound. It was anticipated 

 that appropriate concentrations of azide might inhibit the hydrolytic 

 activity without aff^ecting the transfer activity. These experiments were 

 also unsuccessful. We have recently isolated from the submitochondrial 

 particles a substance which is a potent inhibitor of the ATPase and appears 

 to have no effect on oxidative phosphorylation in the reconstituted system. 



