Glutamine synthetases from rice: purification and preliminary characterization of two forms in leaves and one form in roots

Abstract

A relatively rapid five-step procedure was used in purifying to apparent homogeneity the glutamine synthetase from roots and one form of the enzyme (GS_I) from leaves of rice. The steps were: preparation of crude extracts, ammonium sulfate precipitation, filtration on Sepharose 4B, fractionation on DEAE-Sephadex A25, and affinity chromatography on ADP-Sepharose 4B. The purified protein appeared as a single band on polyacrylamide gel electrophoresis. Leaf GS_I and the second type of leaf glutamine synthetase (GS_II) formed distinct peaks when eluted from DEAE-Sephadex (step 4). The root enzyme and leaf GS_I were similar in all the properties which were examined. Both enzymes bound to ADP-Sepharose, had similar biosynthetic (18 µmol P/img protein/min) and transferase (1324 and 1156 µmol γ-glutamyl hydroxamate/mg protein/min) activities, and the same or nearly the same K_m values for glutamate (2.17 mm), Mg²⁺ (4.5 and 5.0 mm), ATP (286 µm), NH₄⁺ (210 and 135µm), and ADP (3.8 and 5.3 µm). In contrast, leaf GS_II did not bind to ADP-Sepharose and had much higher K_m values for glutamate (8.3 mm), Mg²⁺ (15 mm), NH₄⁺ (684 µm), and ADP (33 µm).