title: An NADP-glutamate dehydrogenase from the green alga Bryopsis maxima. Purification and properties.
abstract: NADP-glutamate dehydrogenase (EC 1.4.1.4:NADP-GDH) was purified to electrophoretic homogeneity from the multinuclear-unicellular green marine alga in Siphomales, Bryopsis maxima, and its properties were examined. M(r) of the undenatured enzyme was 280 kDa, and the enzyme is thought to be a hexamer of 46 kDa subunit protein. Optimum pHs for the reductive amination and oxidative deamination were 7.5 and 8.2-9.0 respectively. The enzyme displayed NADPH/NADH-specific activities with a ratio of 18:1. Apparent K(m) values for 2-oxoglutarate, ammonia, NADPH, glutamate and NADP+ were 3.0, 2.2, 0.03, 3.2 and 0.01 mM respectively. The enzymochemical characteristics of the GDH were studied and compared to those of other species. The B. maxima GDH was insensitive to 5 mM Ca(2+) and to 1 mM EDTA in contrast to higher plant NAD-GDHs. Chemical modifications with DTNB and pCMBS suggested that cysteine residues are essential for the enzymatic activity as in other species GDHs. The GDH was not affected by 1 mM purine nucleotides, suggesting that the enzyme is not allosteric, in contrast to animal NAD(P)-GDHs and fungal NAD-GDHs.
authors.affiliation: Department of Biomolecular Science, Toho University, Chiba, Japan.
authors.forename: R
authors.initials: R
authors.lastname: Inokuchi
authors.forename: T
authors.initials: T
authors.lastname: Itagaki
authors.forename: J T
authors.initials: JT
authors.lastname: Wiskich
authors.forename: K
authors.initials: K
authors.lastname: Nakayama
authors.forename: M
authors.initials: M
authors.lastname: Okada
mesh_terms: D000595:Amino Acid Sequence; D000460:Chlorophyta / Q000201:enzymology*; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D005969:Glutamate Dehydrogenase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry
chemical_list: D046911:Macromolecular Substances; D010446:Peptide Fragments; D005969:Glutamate Dehydrogenase; C061421:glutamate dehydrogenase (NAD(P)+)