MULTIPLE ENZYME SYSTEMS 



information, but it may not always be fruitful at the beginning. 

 One has only to consider the situation in the study of glycolysis. 

 In the earlier work it seems reasonable to assume that fractiona- 

 tion of the soluble extract which converted glucose to ethanol 

 would not have brought about the accumulation of sizable 

 quantities of intermediates, since the over-all reaction is neces- 

 sary for the regeneration of the proper acceptor systems. To be 

 sure, later when some of the details of the mechanism had be- 

 come known, individual steps could be isolated and the products 

 studied. This had to await the finding of intermediates in the 

 reaction sequence, however. In this regard then the old- 

 fashioned careful complete balance study is perhaps the most 

 dependable beginning tool. Time and again advances in such 

 studies arise from a consideration of the balance. The work 

 involved in the development of the quantitative methods may 

 be revolting, but the results are never wasted and are always 

 useful. In many cases the balance can differentiate between 

 two possible mechanisms or may provide evidence for the pres- 

 ence of an intermediate compound. Even when the reaction 

 appears to be straightforward, a balance may change one's 

 ideas. The classic findings of Harden and Young depended on 

 a demonstration of net phosphate uptake during glycolysis. It is 

 important to balance as many of the reactants as possible, since 

 a partial balance can lead to an erroneous picture of the reaction. 



INTERMEDIATES 



The problem always arises in studying a MES whether to 

 try to isolate possible intermediate compounds in the over-all 

 system or to try to fractionate so as to facilitate the accumulation 

 of such compounds. Naturally no single approach can be 

 applied to all problems. When intermediate compounds ac- 

 cumulate fortuitously, their isolation and characterization be- 

 comes an obvious first step. If not, fractionation, the use of 

 inhibitors, or omission of substrates may lead to accumulation 

 of intermediates. In any event it may be necessary to obtain 

 mtermediates by special tricks. Once an in vitro system is 



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