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



Similarly, a 2-hr. dialysis at room temperature resulted in parallel 

 losses of both activities. These results are presented in Table V. The 

 addition of ATP to the dialyzing solution at a final concentration of 

 0-005 ^^ again resulted in considerable protection of both activities. 

 Various salts, for example, ammonium sulphate, ammonium chloride or 

 potassium sulphate, added to the dialyzing medium at concentrations then 

 ten times higher than that of the ATP also prevented to a large extent the loss 

 of both activities. Attempts to reactivate the dialyzed enzyme by the 

 addition of boiled enzyme, cold inactivated enzyme or several known 

 cofactors have been unsuccessful. 



TABLE V 



Protection by ATP Against Dialysis Inactivation of ATPase And Coupling 



Activity 



2-8 mg. of the coupling enzyme were dissolved in 1-5 ml. sucrose-tris-EDTA 

 and divided into three o • 5 ml. aliquots. Dialysis was carried out at room temperature 

 for 2 hr. vs. 0-25 M sucrose-o-oi M tris, pH 7-4. ATP was 0-005 M when added. 

 Assays for coupling activity (P :0) were carried out as described previously [4] 

 with 0-038 mg. of the coupling factor and 0-620 mg. of the particulate fraction per 

 vessel. In the absence of coupling factor the P :0 was 003. ATPase activity was 

 measured with the ATP regenerating system [3]. 



Dialyzing solution P:0 ATPase 



Additional evidence for the single enzyme concept was obtained from 

 an examination of the effect of uncouplers on the two activities. Some of 

 these results are summarized in Table VI. In general it was found that all 

 compounds which affect the ATPase (i.e. either inhibit or stimulate) also 

 uncoupled oxidative phosphorylation. /)-Chloromercuribenzoate, azide, 

 Dicoumarol, dihydrovitamin K^ diphosphate (a water-soluble derivative 

 of vitamin Kj) and triiodothyronine inhibited both the phosphorylation 

 activity and the ATPase activity of the purified factor at concentrations 

 between 5 x 10 ^ and 5 x io~^ m. Dinitrophenol and pentachlorophenol, 

 two potent uncouplers of oxidative phosphorylation, inhibited the phos- 

 phorylation activity and stimulated the ATPase activity. Azide inhibited 

 the ATPase activity to a greater extent in the absence than in the presence 

 of dinitrophenol with the result that the stimulation by dinitrophenol was 

 in effect increased from 50 to 300 or 400",,. The opposite effect was 

 observed with /)-chloromercuribenzoate which completely eliminated the 

 dinitrophenol stimulation. 



