title: Purification and characterization of NADP-dependent glutamate dehydrogenase from the commercial mushroom Agaricus bisporus.
abstract: The nicotinamide adenine dinucleotide phosphate (NADP)-dependent glutamate dehydrogenase (NADP-GDH) of Agaricus bisporus, a key enzyme in ammonia assimilation, was purified to apparent electrophoretic homogeneity with 27% recovery of the initial activity. The molecular weight of the native enzyme was 330 kDa. The enzyme is probably a hexamer, composed of identical subunits of 48 kDa. The isoelectric point of the enzyme was found at pH 4.8. The N-terminus appeared to be blocked. The enzyme was specific for NADP(H). The Km-values were 2.1, 3.2, 0.074, 27.0, and 0.117 mM for ammonia, 2-oxoglutarate, NADPH, L-glutamate, and NADP respectively. The pH optima for the amination and deamination reactions were found to be 7.6 and 9.0, respectively. The temperature optimum was 33 degrees C. The effect of several metabolites on the enzyme's activity was tested. Pyruvate, oxaloacetate, ADP, and ATP showed some inhibitory effect. Divalent cations slightly stimulated the aminating reaction. Antibodies raised against the purified enzyme were able to precipitate NADP-GDH activity from a cell-free extract in an anticatalytic immunoprecipitation test. Analysis of a Western blot showed the antibodies to be specific for NADP-GDH.
authors.affiliation: Department of Microbiology, Faculty of Science, University of Nijmegen, The Netherlands.
authors.forename: J J
authors.initials: JJ
authors.lastname: Baars
authors.forename: H J
authors.initials: HJ
authors.lastname: Op den Camp
authors.forename: A H
authors.initials: AH
authors.lastname: van Hoek
authors.forename: C
authors.initials: C
authors.lastname: van der Drift
authors.forename: L J
authors.initials: LJ
authors.lastname: Van Griensven
authors.forename: J
authors.initials: J
authors.lastname: Visser
authors.forename: G D
authors.initials: GD
authors.lastname: Vogels
mesh_terms: D000227:Adenine Nucleotides / Q000494:pharmacology; D000364:Agaricus / Q000201:enzymology*; D000918:Antibody Specificity; D002264:Carboxylic Acids / Q000494:pharmacology; D018086:Glutamate Dehydrogenase (NADP+) / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight
chemical_list: D000227:Adenine Nucleotides; D002264:Carboxylic Acids; D018086:Glutamate Dehydrogenase (NADP+)