Nicotinamide Adenine Dinucleotide Phosphate-specific Glutamate Dehydrogenase of Neurospora

Abstract 1. An improved procedure, utilizing affinity chromatography in the final step, has been devised for the purification of the NADP-specific glutamate dehydrogenase of Neurospora crassa. 2. The molecular weight of the native protein is 288,400 by sedimentation-equilibrium measurements. In 6 m guanidine HCl and 0.5% mercaptoethanol, the subunit molecular weight is 48,800, consistent with a native molecule composed of six subunits of identical size. 3. The enzymic activity is abolished by reaction with pyridoxal 5'-phosphate, the reaction occurring preferentially at a single lysine residue. Only one fluorescent spot is observed on tryptic peptide maps of the enzyme inactivated with this reagent and reduced with borohydride. 4. All six of the free sulfhydryl groups in each subunit can be carboxymethylated or carboxamidomethylated with no effect on catalytic activity. 5. Treatment of the enzyme with N-ethylmaleimide results in loss of activity, whether or not the sulfhydryl groups have been previously blocked by carboxymethylation. The pKa of the group involved is 7.60 at 34° and 8.70 at 2°. The calculated heat of ionization is 12.6 ± 2.0 Cal per mole. Present evidence indicates that the reaction with N-ethylmaleimide probably involves the same e-amino group as in the reaction with pyridoxal 5'-phosphate.