138 Albert L. Lehninger, et al. 



between (2) and (3). However, if so, the relative sites of action 

 of the uncouphng agencies must be in the sequence shown, 

 regardless of the number of intervening reactions. 



We have recently found it possible to dissociate the enzyme 

 catalysing reaction (3) in soluble form from the digitonin 

 fragments and have purified it substantially by classical 

 fractionation methods. It is quite stable and requires no 

 dialy sable cof actor other than Mg2+. It is thus the first 

 enzyme of the coupling mechanism reported in soluble and 

 purified condition and will provide access to the preceding 

 reactions of energy coupling to the respiratory chain through 

 in vitro reconstruction methods utilizing purified enzyme 

 components. The soluble enzyme is inhibited by PCMB, but 

 not by DNP, dicoumarol, gramicidin, azide, KCl or sucrose. 

 These observations not only confirm the findings on the 

 digitonin fragments, but also provide an unequivocal land- 

 mark for identification of sites of action of uncoupling agents. 



Naturally occurring controlling factors in mitochondria 



There have been frequent suggestions that agents exist in the 

 cell which act as physiological uncoupling agents, which can 

 lower the efficiency of energy coupling and also stimulate 

 respiration by releasing it from its rate-dependence on ADP 

 and phosphate. Thyroxine has been visualized as one such 

 agent and our more recent findings indicate that it causes a 

 "dislocation" of a respiratory carrier, a bound form of DPN 

 (Lehninger, Ray and Schneider, 1959). However, it appears 

 probable that mitochondria themselves must also contain 

 uncoupling and/or respiration-releasing factors, since in 

 fresh mitochondria ADP and phosphate are obligatory for 

 respiration to occur whereas in aged mitochondria neither 

 ADP nor Pj is required for maximum rates of respiration, 

 nor do they significantly aff'ect the rate. Mechanical or 

 chemical disruption of mitochondria also often produces 

 similar transformations from phosphorylating ADP-controlled 

 electron transport to non-phosphorylating respiration inde- 

 pendent of ADP. Such findings pose rather fundamental 



