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J. Biol. Chem., Vol. 279, Issue 16, 16598-16605, April 16, 2004

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Kinetic Properties and Ammonium-dependent Regulation of Cytosolic Isoenzymes of Glutamine Synthetase in Arabidopsis^*

Keiki Ishiyama, Eri Inoue, Akiko Watanabe-Takahashi, Mitsuhiro Obara¶, Tomoyuki Yamaya, and Hideki Takahashi||

From the RIKEN Plant Science Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan and theGraduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai 981-8555, Japan

Glutamine synthetase (GS; EC 6.3.1.2) is a key enzyme of nitrogen assimilation, catalyzing the synthesis of glutamine from ammonium and glutamate. In Arabidopsis, cytosolic GS (GS1) was accumulated in roots when plants were excessively supplied with ammonium; however, the GS activity was controlled at a constant level. The discrepancy between the protein content and enzyme activity of GS1 was attributable to the kinetic properties and expression of four distinct isoenzymes encoded by GLN1;1, GLN1;2, GLN1;3 and GLN1;4, genes that function complementary to each other in Arabidopsis roots. GLN1;2 was the only isoenzyme significantly up-regulated by ammonium, which correlated with the rapid increase in total GS1 protein. GLN1;2 was localized in the vasculature and exhibited low affinities to ammonium (K_m = 2450 ± 150 µM) and glutamate (K_m = 3.8 ± 0.2 mM). The expression of the counterpart vascular tissue-localizing low affinity isoenzyme, GLN1;3, was not stimulated by ammonium; however, the enzyme activity of GLN1;3 was significantly inhibited by a high concentration of glutamate. By contrast, the high affinity isoenzyme, GLN1;1 (K_m for ammonium < 10 µM; K_m for glutamate = 1.1 ± 0.4 mM) was abundantly accumulated in the surface layers of roots during nitrogen limitation and was down-regulated by ammonium excess. GLN1;4 was another high affinity-type GS1 expressed in nitrogen-starved plants but was 10-fold less abundant than GLN1;1. These results suggested that dynamic regulations of high and low affinity GS1 isoenzymes at the levels of mRNA and enzyme activities are dependent on nitrogen availabilities and may contribute to the homeostatic control of glutamine synthesis in Arabidopsis roots.

Received for publication, December 15, 2003 , and in revised form, January 22, 2004.

^* This work was supported in part by Grants-in-aid for Scientific Research 14360035 and 14654160 from the Ministry of Education, Culture, Sports, Science and Technology of Japan and by Core Research for the Evolutional Science and Technology (CREST) of Japan Science and Technology. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ Recipient of a post-doctoral research fellowship from the Japan Society for the Promotion of Science.

^|| To whom correspondence should be addressed. Tel.: 81-45-503-9577; Fax: 81-45-503-9609; E-mail: hideki{at}postman.riken.go.jp.

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