NUCLEOTIDES AND COENZYMES 



(gradient elution). This permits a complete separation of the 

 acid-soluble nucleotides in tissues, and has revealed a complete 

 spectrum of mono-, di-, and tri-phospho derivatives of the 

 various purine and pyrimidine nucleosides. 



The usefulness of such ion-exchange columns cannot be 

 overemphasized. Not only does this method provide a tool for 

 rapidly determining the constituent nucleotides of a given 

 tissue, but it can be applied with equal success to the large-scale 

 isolation of individual nucleotides. It came as a shock to some, 

 and a pleasant surprise to others, to learn that supposedly pure 

 samples of ATP of commercial origin were, in reality, wonder- 

 fully complex mixtures of the other triphosphates, and undoubt- 

 edly many ATP-requiring systems will bear re-evaluation. 

 Initially, participation of guanosine diphosphate in the reaction : 



succinyl CoA + P + ADP > succinate + CoA + ATP (7) 



was overlooked until careful scrutiny of ADP samples by Sanadi 

 and co-workers (37) revealed the presence of the true coenzyme — 

 a situation somewhat reminiscent of the discovery of glucose- 

 1,6-diphosphate as a trace impurity in samples of glucose-1- 

 phosphate. 



During purification a most serious problem is the lability of 

 the coenzyme to chemical degradation, especially at the pyro- 

 phosphate and 7V-glycoside linkages. A corollary cf this prob- 

 lem is the facile migration of phosphate groups. Although 

 phosphate groups can be located to some degree on sugar resi- 

 dues by the periodate titration method, the technique does not 

 distinguish between 2'- and 3 '-isomers. The ready intercon- 

 version of adenosine-2- 'phosphate to the 3 '-isomer through the 

 cyclic 2',3'-diester caused the expenditure of great effort in 

 several laboratories before it was finally established that pure 

 adenylic acid "a" was the 2 '-isomer and "b" the 3 '-isomer, 

 rather than vice versa. 



One other fact should be emphasized at this point, and that 

 is the ease with which nucleotides form cyclic phosphate deriva- 

 tives. Thus, glucose- 1,2-phosphate, adenosine-2 ',3 '-phosphate 



501 



