ATPase in oxidative phosphorylation 245 



observed that incubation of the enzyme at 30' or in the presence of ATP 

 at considerably higher temperatures resuhed in an activation of the enzyme. 

 It was necessary, however, to accumulate more direct evidence that 

 the ATPase and coupling activity were in fact catalytic expressions of a 

 single protein. Compelling evidence in favour of this view was obtained 

 by a number of procedures designed to selectively destroy one of the 

 activities. Invariablv these procedures resulted in a parallel destruction of 

 both activities. The most striking of these parallelisms was noted during 

 the later stages of the purification procedure when the ATPase activity 

 became extremelv unstable. Further investigations revealed that the 

 purified preparation was markedly cold-labile. That is, the activity of the 

 ATPase declined rapidly at ice bath temperature while at room tempera- 

 ture the activitv generallv increased. This rather unusual lability was also 

 displayed by the coupling activity. These results are shown in Fig. i. In 



TABLE IV 



Protection by ATP Against Heat Inactivation of ATPase and 

 Phosphorylation Activity 



A solution of the purified enzyme containing i -6 mg. protein/ml. was divided 

 into approptiate aliquots and heated for 4 min. under the indicated conditions. 

 The ATP, when present, was 4 x 10^ M. Assays for coupling activity (P:0) 

 were carried out as describeci previously [4] with 0-038 mg. of the coupling enzyme 

 and 0-495 mg. of the particulate fraction. In the absence of the coupling enzyme, 

 the P:0 ratio was 0-05. The ATPase assay was carried out in the presence of 

 the ATP regenerating system [3]. 



Pretreatment P : O ATPase 



None 



60^ + ATP 



this experiment, aliquots of the purified protein fraction were preincubated 

 either at o' or 30 for the indicated intervals and then added to the appro- 

 priate assay system. Both assays were carried out at 30 . As may be seen, 

 the rapid rates of inactivation of these two activities at o \\ ere strikingly 

 parallel while at 30 both activities were retained for over 20 hr. The 

 enzyme may be kept, however, at 4" as a suspension in 50",, ammonium 

 sulphate for 3 weeks without appreciable loss in either ATPase or coupling 

 activity. 



Exposure of the purified enzyme to elevated temperatures also failed 

 to achie\e a separation of the two activities. As shown in Table IV, both 

 activities were inactivated at 60 to a similar extent and were completely 

 protected by ATP. 



