title: The isoenzyme 7 of tobacco NAD(H)-dependent glutamate dehydrogenase exhibits high deaminating and low aminating activities in vivo.
abstract: Following the discovery of glutamine synthetase/glutamate (Glu) synthase, the physiological roles of Glu dehydrogenase (GDH) in nitrogen metabolism in plants remain obscure and is the subject of considerable controversy. Recently, transgenics were used to overexpress the gene encoding for the beta-subunit polypeptide of GDH, resulting in the GDH-isoenzyme 1 deaminating in vivo Glu. In this work, we present transgenic tobacco (Nicotiana tabacum) plants overexpressing the plant gdh gene encoding for the alpha-subunit polypeptide of GDH. The levels of transcript correlated well with the levels of total GDH protein, the alpha-subunit polypeptide, and the abundance of GDH-anionic isoenzymes. Assays of transgenic plant extracts revealed high in vitro aminating and low deaminating activities. However, gas chromatography/mass spectrometry analysis of the metabolic fate of (15)NH(4) or [(15)N]Glu revealed that GDH-isoenzyme 7 mostly deaminates Glu and also exhibits low ammonium assimilating activity. These and previous results firmly establish the direction of the reactions catalyzed by the anionic and cationic isoenzymes of GDH in vivo under normal growth conditions and reveal a paradox between the in vitro and in vivo enzyme activities.
authors.affiliation: Department of Biology, University of Crete, 71409 Heraklion, Greece.
authors.forename: Damianos S
authors.initials: DS
authors.lastname: Skopelitis
authors.forename: Nikolaos V
authors.initials: NV
authors.lastname: Paranychianakis
authors.forename: Antonios
authors.initials: A
authors.lastname: Kouvarakis
authors.forename: Apostolis
authors.initials: A
authors.lastname: Spyros
authors.forename: Euripides G
authors.initials: EG
authors.lastname: Stephanou
authors.forename: Kalliopi A
authors.initials: KA
authors.lastname: Roubelakis-Angelakis
mesh_terms: D000838:Anions / Q000378:metabolism; D008401:Gas Chromatography-Mass Spectrometry; D005969:Glutamate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism*; D007527:Isoenzymes / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D030821:Plants, Genetically Modified / Q000378:metabolism; D021122:Protein Subunits / Q000378:metabolism; D014026:Nicotiana / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic; D027843:Vitis / Q000235:genetics*
chemical_list: D000838:Anions; D007527:Isoenzymes; D021122:Protein Subunits; D018698:Glutamic Acid; D005969:Glutamate Dehydrogenase