PURIFICATION AND CHARACTERIZATION OF THE NAD-DEPENDENT GLUTAMATE-DEHYDROGENASE IN THE ECTOMYCORRHIZAL FUNGUS LACCARIA BICOLOR (MAIRE) ORTON

The NAD-dependent glutamate dehydrogenase (GDH) (EC 1.4.1.2) from Lacc aria bicolor was purified 410-fold to apparent electrophoretic homogen eity with a 40% recovery through a three-step procedure involving ammo nium sulfate precipitation, anion-exchange chromatography on DEAE-Tris acryl, and gel filtration, The molecular weight of the native enzyme d etermined by gel filtration was 470 kDa, whereas sodium dodecyl sulfat e-polyacrylamide gel electrophoresis gave rise to a single band of 116 kDa, suggesting that the enzyme is composed of four identical subunit s, The enzyme was specific for NAD(H). The pH optima were 7.4 and 8.8 for the amination and deamination reactions, respectively, The enzyme was found to be highly unstable, with virtually no activity after 20 d ays at -75 degrees C, 4 days at 4 degrees C, and 1 h at 50 degrees C, The addition of ammonium sulfate improved greatly the stability of the enzyme and full activity was still observed after several months at - 75 degrees C, NAD-GDH activity was stimulated by Ca2+ and Mg2+ but str ongly inhibited by Cu2+ and slightly by the nucleotides AMP, ADP, and ATP, The Michaelis constants for NAD, NADH, 2-oxoglutarate, and ammoni um were 282 mu M, 89 mu M, 1.35 mM, and 37 mM, respectively. The enzym e had a negative cooperativity for glutamate (Hill number of 0.3), and its K-m value increased from 0.24 to 3.6 mM when the glutamate concen tration exceeded 1 mM, These affinity constants of the substrates, com pared with those of the NADP-GDH of the fungus, suggest that the NAD-G DH is mainly involved in the catabolism of glutamate, while the NADP-G DH is involved in the catalysis of this amino acid. (C) 1997 Academic Press.