Characterization of tomato leaf glutamine synthetase

Glutamine synthetase was purified to apparent homogeneity from the leaves of light-grown tomato plants. The purification steps involved ammonium sulphate precipitation (40–60%), adsorption by hydroxyapatite, DEAE Sephadex ionic exchange chromatography, and Sephacryl S 300 gel filtration. Only one glutamine synthetase form was found, with K_m-values of 5.5 mM, 0.8 mM and 0.4 mM for glutamate, ATP and ammonium, respectively. The in vitro activity depended on Mg²⁺ or Mn²⁺ concentrations with positive cooperativity. Enzyme assay pH optima were 7.2 for synthetase, and 6.0 for transferase. The activation energy of the enzymatic reaction was 33.2 kJ/mol. ADP appeared to be a competitive inhibitor with a K_i-value of 0.4 mM. Enzyme inhibition was also produced by aspartate, alanine and phosphoserine.