Anti-Glutamine Synthetase Anticorps (A101718)

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.

In maize, a small multigene family encodes the cytosolic isoforms of glutamine synthetase (GS), and five cDNAs, designated pGS1a, pGS1b, pGS1c, pGS1d, and pGS1e, have been cloned (Sakakibara, H., Kawabata, S., Takahashi, H., Hase, T., and Sugiyama, T. (1992) Plant Cell Physiol. 33, 49-58; Li, M., Villemur, R., Hussey, P. J., Silflow, C. D., Gantt, J. S., and Snustad, D. P. (1993) Plant Mol. Biol. 23, 401-407). This report describes the identification and enzymatic characterization of the cytosolic isoforms of GS in maize roots, namely GS1 and GSr. The purified isoforms, as well as recombinant enzymes that had been overexpressed in Escherichia coli, were analyzed by capillary liquid chromatography/electrospray ionization-mass spectrometry, and GS1 and GSr were identified as the products of the GS1a/GS1b and GS1c/GS1d genes, respectively. Upon the addition of ammonia to the culture medium, significant amounts of GSr accumulated and a preferential increase in GS synthetase activity, as compared to GS transferase activity, was found in the root extract. Assays with the purified recombinant enzymes confirmed that the specific biosynthetic and synthetase activities of GSr were 1.6-fold higher than those of GS1. Marked differences in stability were also found between the two isoforms: GSr was more sensitive to heat than GS1 and octameric aggregates of the subunits of GSr were easily dissociated to monomers than those of GS1 at low concentrations of Mn2+ and Mg2+ ions. These characteristics of the ammonia-induced isoform of GS seem to be physiologically important for the primary assimilation of external ammonia by roots.