title: Multiple complexes of nitrogen assimilatory enzymes in spinach chloroplasts: possible mechanisms for the regulation of enzyme function.
abstract: Assimilation of nitrogen is an essential biological process for plant growth and productivity. Here we show that three chloroplast enzymes involved in nitrogen assimilation, glutamate synthase (GOGAT), nitrite reductase (NiR) and glutamine synthetase (GS), separately assemble into distinct protein complexes in spinach chloroplasts, as analyzed by western blots under blue native electrophoresis (BN-PAGE). GOGAT and NiR were present not only as monomers, but also as novel complexes with a discrete size (730 kDa) and multiple sizes (>120 kDa), respectively, in the stromal fraction of chloroplasts. These complexes showed the same mobility as each monomer on two-dimensional (2D) SDS-PAGE after BN-PAGE. The 730 kDa complex containing GOGAT dissociated into monomers, and multiple complexes of NiR reversibly converted into monomers, in response to the changes in the pH of the stromal solvent. On the other hand, the bands detected by anti-GS antibody were present not only in stroma as a conventional decameric holoenzyme complex of 420 kDa, but also in thylakoids as a novel complex of 560 kDa. The polypeptide in the 560 kDa complex showed slower mobility than that of the 420 kDa complex on the 2D SDS-PAGE, implying the assembly of distinct GS isoforms or a post-translational modification of the same GS protein. The function of these multiple complexes was evaluated by in-gel GS activity under native conditions and by the binding ability of NiR and GOGAT with their physiological electron donor, ferredoxin. The results indicate that these multiplicities in size and localization of the three nitrogen assimilatory enzymes may be involved in the physiological regulation of their enzyme function, in a similar way as recently described cases of carbon assimilatory enzymes.
authors.affiliation: Institute for Protein Research, Osaka University, Suita, Osaka, Japan.
authors.forename: Yoko
authors.initials: Y
authors.lastname: Kimata-Ariga
authors.affiliation: Institute for Protein Research, Osaka University, Suita, Osaka, Japan.
authors.forename: Toshiharu
authors.initials: T
authors.lastname: Hase
mesh_terms: D002736:Chloroplasts / Q000201:enzymology / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D009584:Nitrogen / Q000378:metabolism*; D018724:Spinacia oleracea / Q000201:enzymology / Q000378:metabolism*
chemical_list: D009584:Nitrogen