Ammonium assimilation in bryophytes. L-glutamine synthetase from Sphagnum fallax

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 (GSI) 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. GS(1) activity could be measured from pH 6.8 to 8.6 in 50 mM imidazole buffer, with a broad optimum between pH 7.2 and 8.0. The K-m values were 2.5 mM, 0.5 mM and 0.5 mM for L-glutamate, ammonium and ATP, respectively. The enzyme was inhibited by more than 10 mM ammonium or glutamate. The incorporation of (NH4+)-N-15 into amino acids was observed in vivo using N-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.