MULTIPLE ENZYME SYSTEMS 



activity and the total activity the actual quantity of compound 

 can be calculated, these data are very useful. Such data have 

 been reported in the in vitro system synthesizing IMP (12). 



Certain problems arise in employing isotopic compounds to 

 study a MES. For example consider the following: 



A* . B , C 



where the equilibria and rates of reaction are such that B does 

 not accumulate. If we start with labeled A, in the absence of 

 B or C, then C will become labeled with time, and the specific 

 activity of C will approach that of A at time zero. If, however, 

 a pool of unlabeled C is added at time zero, the final specific 

 activity of both A and C will be much lower. It follows that 

 when a high concentration of C is present, the rate of incorpora- 

 tion of labeled A is not a true measure of C synthesis but repre- 

 sents a minimal value since A is being diluted by C. This has 

 its practical significance where one attempts to demonstrate 

 exchange reactions. B can be responsible for a significant dilution 

 even though it does not accumulate in quantity. For example, 

 if B is synthesized from a source other than A and G and is in 

 turn converted to another product, i.e., it is turning over rapidly 

 independently of the reaction ABC, a dilution of the radioactive 

 atoms will occur. 



If the reaction B — » C is essentially irreversible, a somewhat 

 diff'erent situation arises. In the system A* ;;^ B — > C, where 

 B -^ C is limiting because of the absence of a cofactor, B should 

 accumulate approximately in accord with the equilibrium con- 

 stant, B/A. Suppose the rate B ^ C is increased by adding 

 the cofactor. Then A ;=± B is drawn towards B and more C will 

 be formed. But the total radioactivity in B should remain 

 essentially unchanged as long as the concentration of A is not 

 limiting. 



The addition of large levels of a suspected product or inter- 

 mediate to determine whether a reaction is reversible is a 

 standard practice. If the rate of substrate disappearance is 

 lessened, this is taken as some evidence that the reaction is 



557 



