Comparative characterization of glutamine synthetase isoforms from Canavalia lineata in biochemical and immunological aspects

Two isoforms of glutamine synthetase were identified from Canavalia lineata leaves and two forms (designated GS1 and GS2) showed different properties. The molecular weight estimated by gel filtration was 340 kDa for the GS1 and 407 kDa for the GS2. The optimum pH of the GS2 (pH 8.0) was somewhat higher than that of the GS1 (pH 7.5), while, the activation energies and the optimum temperatures of the two isoforms were similar. The inhibition patterns of the isoforms by methionine sulfoximine were different from each other: The competitive inhibition for the GS1 with a very low K-is value of 0.033 mM was compared to the non-competitive inhibition for the GS2 with a K-is value of 0.27 mM and a K-ii value of 0.17 mM. The result of the initial velocity studies for the isoenzymes was compatible with a sequential-random kinetic mechanism. The kinetic parameters at 35 degrees C and pH 7.8 were K-Glu = 5.29 mM, K-ATP = 0.24 mM, and K-Hydroxylamine = 0.07 mM for the GS1 and K-Glu = 2.14 mM, K-ATP = 0.57 mM, and K-Hydroxylamine = 0.09 mM for the GS2. The Viola and Cleland's method used in this kinetic analysis was more exact and more acceptable than other methods used until now to analyze the glutamine synthetase. The molecular weight of GS2 purified with Cibacron blue dye chromatography was 42 kDa and the anti-GS2 antibody could detect three different forms of GS subunit in different organs. (C) 1998 Elsevier Science Ireland Ltd. All rights reserved.