Ammonium assimilation in bryophytes. l-glutamine synthetase from Sphagnum fallax |
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Authors: | Stefan Kahl Jóska Gerendás Volker Heeschen R George Ratcliffe Hansjörg Rudolph |
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Institution: | Botanisches Inst. der Christian-Albrechts-Univ. zu Kiel, Biologiezentrum, Olshausenstraβe 20–40, D–24098, Germany;Inst. für Pflanzenernährung und Bodenkunde der Christian-Albrechts-Univ. zu Kiel, Olshausenstraβe 40, D–24098 Kiel, Germany;Dept of Plant Sciences, Univ. of Oxford, South Parks Road, Oxford, OXI 3RB, UK. |
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Abstract: | Cytosolic and plastidic l -glutamine synthetase (EC 6.3.1.2) isoenzymes from Sphagnum fallax Klinggr. (Klinggr. clone 1) were separated by size-exclusion and ion exchange chromatography. The cytosolic enzyme (GS1) was purified to apparent electrophoretic homogeneity. The native enzyme had a molecular mass of 390 ± 20 kDa as estimated by gel filtration and was apparently composed of 8 subunits with molecular masses of 48 kDa. GS1 activity could be measured from pH 6.8 to 8.6 in 50 m M imidazole buffer, with a broad optimum between pH 7.2 and 8.0. The Km values were 2.5 m M , 0.5 m M and 0.5 m M for l -glutamate, ammonium and ATP, respectively. The enzyme was inhibited by more than 10 m M ammonium or glutamate. The incorporation of 15NH4+ into amino acids was observed in vivo using 15 NMR. Label from ammonium was first detected in the amide N of glutamine, and only subsequently in the amino N of glutamate. Moreover, no assimilation was detected in the presence of the specific GS inhibitor methionine sulfoximine. These observations are consistent with a dominant role for GS in the assimilation of ammonium in Sphagnum . |
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Keywords: | Bryophytes compartmentalization l-glutamine synthetase isoenzymes 15N NMR purification Sphagnum fallax |
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