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Purification and properties of glutamine synthetase from Douglas fir roots
Author(s) -
Bedell JeanPhilippe,
Chalot Michel,
Brun Annick,
Botton Bernard
Publication year - 1995
Publication title -
physiologia plantarum
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.351
H-Index - 146
eISSN - 1399-3054
pISSN - 0031-9317
DOI - 10.1111/j.1399-3054.1995.tb00973.x
Subject(s) - glutamine synthetase , size exclusion chromatography , enzyme , glutamine , chemistry , chromatography , molecular mass , cooperativity , ammonium , polyacrylamide gel electrophoresis , affinity chromatography , specific activity , biochemistry , transferase , enzyme assay , gel electrophoresis , glycine , amino acid , organic chemistry
Glutamine synthetase (GS. EC 6.3.1.2) was purified to apparent electrophoretic homogeneity from roots of Pseudotsuga menziesii (Mirb) Franco by a three‐step procedure involving diethylaminoethyl (DEAE)‐Trisacryl chromatography, affinity chromatography on Matrex Gel Red A. and preparative polyacrylamide gel electrophoresis. The enzyme was purified 40‐fold with a 16% recovery. The native enzyme had a molecular mass of 460 ± 5 kDa as estimated by gel filtration, interpolation of the Ferguson plots and non‐denaturing gradient‐PAGE. It was composed of two different subunits of 54 and 64 kDa. Affinity constants for glutamate (Glu), glutamine (Gln), ATP and ADP were 2.6, 10.5, 0.5 and 0.083 m M . respectively. The enzyme exhibited a negative cooperativity for ammonium (Hill number of 0.7) with two K m values which were 11 and 75 μ M in the presence of ammonium concentrations lower and higher than 1.3 m M , respectively. Glycine and ADP appeared as potential inhibitors of the GS activity. The optimum pH values were 7.2 and 7.6 for the transferase and the biosynthetic assays, respectively. The enzyme lost 30% of its activity within 25 days of storage at 4°C. The optimum temperatures of activity were 40°C and 45°C for the transferase and bio‐synthetic activities, respectively.