Kinetic and equilibrium studies on crystalline 1-glutamic acid dehydrogenase.

The spectrophotometric procedure for determining the specific activity of the enzyme and the preparation of reagents employed in the assay have been described previously (3). DPiV-Commercial preparations of DPN (60 per cent) were used in routine assays. For critical experiments, commercial DPN was further purified by several adsorptions on Dowex 1 formate columns by the method of Kornberg and Pricer (6). The dried DPN was assayed by the enzymatic method of Bonnichsen (7), in which the crystalline yeast alcohol enzyme (8) was employed rather than the liver enzyme. The best preparations were 84 per cent pure as determined by the formula, micromoles of DPN per cc. = (optical density)/6.22 (9), in which optical density is the net increase in optical density at 340 mp. This formula was also used in calculating the purity of TPN and desamino DPN. TPN-TPN prepared by the method of Kornberg and Horecker (10)