68 E. C. Slater and W. C. Hulsmann 



freshly prepared liver mitochondria and the ATPase of aged 

 mitochondria, or mitochondrial fragments are: 



(i) The Mg2+ requirement of aged mitochondria, which 

 could be explained by loss of Mg^^ from the mito- 

 chondria during fragmentation. 



(ii) Between pH's 6 and 8, fluoride inhibits the DNP- 

 stimulated ATPase much more than the system in aged 

 mitochondria (Mj^ers and Slater, 19576). This could 

 be explained by assuming that the reactions (3a) and 

 (5) involving X-^-'Ig and X^Ig are sensitive to 

 fluoride, but the hydrolysis of X '^ P (or I '^ P) in 

 aged mitochondria is insensitive. The sensitivity to 

 fluoride of the ^^Pi-ATP exchange reaction (Hiilsmann, 

 1958, unpublished) is in agreement with this explana- 

 tion. 



(iii) Na2S204 has no effect on the ATPase in freshly prepared 

 mitochondria, but stimulates (with peak of activity at 

 pH 7-4) in aged mitochondria. On the basis of the 

 reaction schemes given above, it could be concluded 

 that Na2S204 stimulates the hydrolysis of X ^' I2 (and 

 perhaps also X ^--^ I3) in aged mitochondria. 



(iv) 10~^ M-DNP inhibits the 6-3 enzyme in fresh mito- 

 chondria, but has little effect on aged mitochondria. 

 A possible explanation is that high concentrations of 

 DNP inhibit the hydrolysis of X '^ I3 (promoted by 

 lower concentrations), but have no effect on the 

 hydrolysis of X '-^ P. 



Polls and Shmukler (1957) and Pullman and Racker (1956) 

 showed that it was possible to isolate from aged mitochondria 

 a substance which uncoupled oxidative phosphorylation in 

 fresh mitochondria. Polls and Shmukler (1957) isolated the 

 naturally occurring uncoupling agent from liver mitochondria 

 and purified it. They found that the purified preparation had 

 the spectrum of a haem compound with only one band at 

 410 m(jL when oxidized and 422 m(x when reduced. The reduced 

 form combined with CO, with a slight shift to 418 m^x, and 



