THE CRYSTALLIZATION AND CHARACTERIZATION OF l-GLUTAMIC ACID DEHYDROGENASE

The wide-spread occurrence of the enzyme, L-glutamic acid dehydrogenase, which catalyzes the reaction L-Glutamate + coenzyme + a-ketoglutarate + NH,+ + reduced coenzyme + H+ in bacteria (1, 2), yeast (3, 4), plants (4-6), and mammalian tissues (7, 8) suggests that this enzyme might play an important physiological r6le in nature, possibly in the fixation of ammonia within the cell ( 7) or in hydrogen transport (9).Inasmuch as previous studies on the properties of this enzyme were carried out with impure enzyme preparations, and since conflicting reports concerning the pH optimum (7, 8, 10) and coenzyme specificity (2, 7, 8, 10-12) of the beef liver enzyme have appeared in the literature, an extensive investigation of the properties of the highly purified enzyme was undertaken.The present paper deals with the crystallization of glutamic dehydrogenase and the determination of its physical properties.A brief report (13) of the crystallization procedure has been published previous1y.lEXPERIMENTAL Reagents and Methods ~/3 L-potassium glutamate.Glutamic acid (Eastman Kodak) was suspended in 0.2 M potassium phosphate and neutralized to pH 7.7 with KOH.DPS. 60 per cent pure DPN (diphosphopyridine nucleotide) was made up in 0.2 M potassium phosphate just before use at a concentration of 1 mg.per cc.and neutralized to pH 7.7.The solution may be frozen for short periods without an appreciable loss of activity.