PURIFICATION AND PROPERTIES OF GLUTAMINE-SYNTHETASE FROM DOUGLAS-FIR ROOTS

Glutamine synthetase (GS, EC 6.3.1.2) was purified to apparent electrophoretic homogeneity from roots of Pseudotsuga menziesii (Mirb) France 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 mM, respectively. The enzyme exhibited a negative cooperativity for ammonium (Hill number of 0.7) with two K-m values which were 11 and 75 mu M in the presence of ammonium concentrations lower and higher than 1.3 mM, 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 degrees C. The optimum temperatures of activity were 40 degrees C and 45 degrees C for the transferase and biosynthetic activities, respectively.