COMPONENTS OF THE ENERGY-COUPLING MECHANISM 35 



While this represents the simplest statement, as will be seen it is possible 

 that one or more additional intermediate reactions may also occur. The 

 above sequence accounts for the finding that ADP is necessary for m- 

 corporation of P, into ATP and that P, is not necessary for incorporation 

 of ADP into ATP [2]. It is suggested that this basic mechanism occurs at 

 all three phosphorylation sites of the respiratory chain, but it is not vet 

 known to what extent each of the three sites contributes to the overall 

 rates of the partial reactions. The outline of the reaction pattern described 

 here is in general consistent with most experimental observations, but 

 there have been some difi^erences in interpretation which are fuUv out- 

 lined by Slater [6]. The value of any hypothesis is the fruitfulness of 

 experimentation which it may suggest. 



Separation of the ATP-ADP exchange enzyme 



It was found that the relatively stable enzvme catalyzing the ATP- 

 ADP exchange could be extracted from acetone powders of digitonin 

 fragments and of mitochondria in soluble, highly active form and in 

 nearly complete yield [16]. It has now been purified approximatelv i Re- 

 fold by Dr. Charles L. Wadkins, using ammonium sulphate fractionation 

 and chromatography on cellulose columns. While a minor component is 

 still present, preliminary examination indicates that the protein is of 

 relatively small molecular weight and that it is free of lipid. The highly 

 purified enzyme requires Alg^^ or AIn~^ for activity, is quite stable to 

 dialysis and storage, and is reversibly inhibited by /i-chloromercuri- 

 benzene sulphonate (PCMB). It has been assayed for activity in promoting 

 other phosphate-transferring reactions which are known to bring about 

 ATP-ADP exchanges, such as myokinase and protein phosphokinase, but 

 such activities are absent. In addition the enzyme does not show ATP-P^^'- 

 exchange activity or ATP-ase activity, in the presence or absence of DNP. 

 The enzyme is not identical with that described by Chiga and Plant [17] 

 who have obtained a highly purified enzyme from heart mitochondria 

 catalyzing both the ATP-P,^- exchange and ATP ADP exchange and 

 which is most active with Mn "^ '^. 



Recombination of soluble ATP ADP exchange enzyme with 



digitonin fragments 



The soluble form of the ATP ADP exchange enzyme is completely 

 insensitive to dinitrophenol. In this form it therefore possesses no dis- 

 tinctive characteristics which identify it as a portion of the energy-coupling 

 machinery of oxidative phosphorylation. Experimental approaches were 

 therefore taken to establish more firmly the relevance of this enzyme in 



