VOL. 12 (1953) REACTIONS OF ADENOSINE AND INOSINE PHOSPHATES I79 



DISCUSSION 



The observation that ITP becomes radioactive when added together with radio- 

 active ATP to striated muscle or intestinal mucosa could be accounted for by the anaero- 

 bic reaction 



ATP + IDP — > ADP + ITP. (i) 



The simultaneous fall of the specific activity of ATP phosphorus in suspensions of in- 

 testinal mucosa suggests that the reaction is reversible. The incorporation of radio- 

 active inorganic phosphate could be explained by oxidative phosphorylation leading 

 to radioactive ATP, followed by reaction (i). 



The nature of the enzymes which may catalyse reaction (i) is unsettled. The reaction 

 is not catalysed by myokinase, which transfers phosphate groups from ATP to AMP, 

 and from one ADP molecule to another. As several hydrolytic enzymes, including phos- 

 phatases (AxELROD^^a. Meyerhof AND Green^^), are known to transfer specific radicals 

 either to water (in which case they hydrolyse) or to other hydroxy compounds (in which 

 case they transfer) it is conceivable that the transfer of phosphate is brought about by 

 one of the hydrolytic ATPase. But three of these which were tested - the myosin ATPase, 

 the soluble ATPase of Kielley and Meyerhof and purified alkaline phosphatase fail 

 to catalyse reaction (i). 



Green and Meyerhof^* noted that crude preparations of semen phosphatase 

 transfer phosphate from ATP to glycerol but that purification of the enzyme reduces 

 the ability to transfer ATP phosphorus. At the same time transfer from acetyl phosphate 

 and /)-nitrophenyl phosphate was not affected by purification. The authors deduced 

 the presence of a phosphokinase specific for ATP. If ATPases play a role in the transfer 

 reaction described in this paper they must be supplemented by a phosphokinase. 

 It is also possible that a phosphokinase alone catalyses a phosphate transfer between 

 ATP and ITP. 



The analogy of reaction (i) with the interaction between ATP and creatine raises the 

 question whether ITP, like creatine phosphate, is a storage form of phosphate bond ener- 

 gy. The methods described in this paper have been used by Oljver^^ to measure the 

 concentration of inosine phosphates in various animal tissues, but no appreciable quan- 

 tity {i.e. less than o.i ju,g P per g tissue) was found in frog muscle if the metabolic 

 activities of the tissue were stopped by treatment with dry ice within seconds after 

 removal from the animal (see also Lohmann^^). 



Experiments of Wajzer and Nekhorocheff^' and of Weil-Malherbe^^ suggest 

 that animal tissues can convert IMP into ATP. It is feasible that reaction (i) is a step 

 in this synthesis. 



SUMMARY 



1. A paper chromatographic procedure for the separation of the 5- mono-, di-, and tri-phosphates 

 of adenosine and inosine is described. 



2. The terminal pliosphate group of added ITP becomes radioactive at about the same rate 

 as the two terminal phosphate groups of added ATP when respiring suspensions of pigeon breast 

 muscle are incubated with ^2P04. 



3. ITP also becomes radioactive when incubated anaerobically with suspensions of muscle or 

 intestinal mucosa containing ^^P labelled ATP. 



4. Purified myokinase, myosin ATPase, or soluble ATPase do not transfer phosphate from 

 ATP to inosine phosphates. The two latter enzymes hydrolyse both ATP and ITP, but when the two 

 substrates are added together the rate of hydrolysis is lower than the sum of the rates of reaction found 

 when the substrates are added singly. 



References p. 180. 



