Molecular identification and characterization of cytosolic isoforms of glutamine synthetase in maize roots.
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.
Sakakibara
H
H
Department of Applied Biological Sciences, School of Agricultural Sciences, Nagoya University, Nagoya 464-01, Japan. sakaki@nuagr1.agr.nagoya-u.ac.jp
Shimizu
H
H
Hase
T
T
Yamazaki
Y
Y
Takao
T
T
Shimonishi
Y
Y
Sugiyama
T
T
D000641:Ammonia / Q000494:pharmacology; D003001:Cloning, Molecular; D003600:Cytosol / Q000201:enzymology; D018076:DNA, Complementary; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005974:Glutamate-Ammonia Ligase / Q000235:genetics* / Q000378:metabolism; D006358:Hot Temperature; D007527:Isoenzymes / Q000235:genetics* / Q000378:metabolism; D008274:Magnesium; D008345:Manganese; D018517:Plant Roots / Q000187:drug effects / Q000201:enzymology*; D003313:Zea mays / Q000187:drug effects / Q000201:enzymology*
D018076:DNA, Complementary; D007527:Isoenzymes; D008345:Manganese; D000641:Ammonia; D005974:Glutamate-Ammonia Ligase; D008274:Magnesium