MULTIPLE ENZYME SYSTEMS 



reaction rates may be very slow, and coupling will not appear to 

 have been effected. 



Usually, however, the coupling system may be chosen on 

 the basis of its thermodynamic characteristics. Where a single 

 reaction is being studied, it is generally necessary to couple the 

 system with purified enzymes to prevent an introduction of 

 extraneous reactions. When crude extracts carrying out a 

 multi-enzyme synthesis are being studied, it may be unnecessary 

 to couple the reaction with such a pure enzyme. Frequently 

 the mere addition of the proper substrates and cofactors will 

 make use of the enzymes in the extract, and the proper coupling 

 can be effected with the system being investigated. For ex- 

 ample, an ammonium sulfate fraction of muscle extract (24) 

 may be employed to catalyze the regeneration of ATP with 3- 

 phosphoglycerate as the substrate, but liver extracts carry out 

 this series of reactions very well. By using ATP at low concen- 

 trations and by using a regeneration system, interfering apyrases 

 may be partially circumvented and reasonable phosphate bal- 

 ances effected. As a source of DPNH or of TPNH glucose-6- 

 phosphate is frequently added. Ammonium salts and a- 

 ketoglutarate in the presence of glutamic acid dehydrogenase 

 may act as a good acceptor of electrons from either TPNH or 

 DPNH providing the Eq of the reaction in question is correct. 

 Often, however, the use of pyridine nucleotides as coupled 

 acceptor compounds is beset with diflEiculties. 



High concentrations of extract frequently are required to 

 study complex reactions in crude systems. This appears to be 

 essential because one of the reactions in the chain or one of the 

 coupled reactions is limiting. Therefore it may not be possible 

 to follow the oxidation or reduction of pyridine nucleotides by 

 changes in absorption at 340 mju because the blank may be pro- 

 hibitive. It may be possible to couple the DPN system to other 

 acceptor systems having absorption maxima at longer wave- 

 lengths. However, this procedure can have its difficulties also. 

 Substrate quantities of cytochrome c and DPNH cytochrome c 

 reductase (22) or TPNH cytochrome c reductase (16) provide 



543 



