40 ALBERT L. LEHNINGER 



given above. M-factor therefore could serve as an intermediate enzyme in 

 the respiratory energy coupUng sequence, which is capable of transferring 

 high energy groups from the coupled carrier to the terminal enzyme E 

 catalyzing the ATP-ADP exchange reaction. This possibility is being 

 examined directly (a) by studying the participation of M-factor in the 

 binding of external soluble ATP-ADP exchange enzyme to the presumably 

 empty sites in digitonin particles, to determine the sequence and stoi- 

 chiometry of rebinding, and (b) by examination of complex formation 

 between M-factor protein and the ATP-ADP exchange enzyme by 

 physical methods and by kinetic approaches. Recently we have found that 

 when the soluble ATP-ADP exchange enzyme is more highly purified, 

 it no longer can "recombine" with digitonin fragments to restore DNP- 

 sensitivity. It appears possible that purification has removed a factor 

 necessary for "recombination" and work is in progress to determine 

 whether M-factor is involved in binding soluble ATP-ADP exchange 

 enzyme. 



Relationship to other soluble factors supporting oxidative 

 phosphorylation 



While many investigators have observed that soluble protein fractions, 

 particularly from bacterial extracts, can increase the P : O ratio of res- 

 piratory chain preparations (cf. [22, 23]), in general, little is known of 

 the enzymic capabilities of such soluble fractions and the contribution 

 they make to the overall coupling mechanism. Similarly the protein 

 fraction isolated by Titchener and Linnane from beef heart mitochondria 

 [24], which increases the P : O ratio of pretreated beef heart particles, is 

 of relatively unknown enzymic competence. However, the important 

 work of Pullman, Penefsky, and Packer [25] has shown that a highly 

 purified soluble enzyme catalyzing DNP-stimulated ATP-ase in the 

 presence of Mg + +, increases the P : O ratio of mechanically disrupted 

 beef heart mitochondria. This factor, which shows extraordinary lability 

 to cold, does not catalyze the ATP-Pj^^ exchange reaction or an ATP-ADP 

 exchange reaction. While this soluble ATP-ase seems not to be identical 

 with either our ATP-ADP exchange enzyme or the M-factor described 

 above, at least on superficial comparison of properties, yet it cannot be 

 excluded that there are elements of identity. It is possible for example 

 that a complex of the ATP-ADP exchange enzyme and M-factor may be 

 equivalent to the Pullman ATP-ase, at least in some respects. In any case 

 further development of both lines of work and comparison of the findings 

 should be of great importance. It must be recalled that there are three 

 phosphorylation sites in the respiratory chain. While it is comforting to 

 think that all three operate by the same mechanism, this need not be the 



