G. ROBERT GREENBERG 



tionation of the system would eventually show E to be a side 

 product. 



Specific radioactivity measurements do not in themselves 

 prove that a compound is an intermediate. Thus in studying 

 the conversion of hypoxanthine to inosinic acid the author (12) 

 found the following specific activities (counts/^umole) after 

 incubation of C^*-hypoxanthine with pigeon liver for 21 minutes: 

 hypoxanthine, 4500; inosine, 2150; and inosinic acid, 1380. 

 Furthermore inosine appeared to be a better precursor of IMP 

 than hypoxanthine was. Accordingly it was considered rea- 

 sonable to suggest that the reaction mechanism was via inosine 

 perhaps as follows : 



ATP 



hypoxanthine + ribose-1 -phosphate > inosine > IMP 



However, Buchanan and co-workers (30) have fractionated this 

 system and their results together with the discovery of PRPP* 

 (18) provide evidence that the following holds: 



(1) 

 Inosine , _ + |ATP 



> 



PRPP 



I at: 



R-5-P 



Hypoxanthine was more rapidly equilibrated with inosine (1) 

 than it was converted to IMP (2), and therefore inosine had 

 shown an intermediate specific activity in our experiments. 



Specific activity measurements against time may give more 

 positive indication that a compound is an intermediate. But 

 even when the compounds follow the typical precursor-product 

 curves, the results are not unequivocal. More adequate evi- 

 dence is obtained when in addition the total activity of each 

 fraction is recorded. This allows one to observe that a net 

 quantity of C^^ activity has been transferred from one compound 

 to another over a period of time. Since from the specific 



* Dr. J. M. Buchanan, private communication. 



556 



