Résumé
A modified procedure for purification of glutamine synthetase [L-glutamate: ammonia ligase (ADP-forming)] from N2-fixing cyanobacterium N. muscorum, to homogeneity is described using DEAE-Sephadex and Blue-Sepharose affinity chromatography. Specific activities of the purified enzyme in biosynthetic and transferase assays were 8.5 and 28 mumole product formed min-1 mg-1 protein. Apparent molecular mass of native GS enzyme was about 610 kDa as estimated by gel filtration. On SDS-PAGE the enzyme protein migrated as single band with molecular weight of 51 kDa. Apparent Michaelis Menten constant (Km) for glutamate, glutamine, ATP and ammonium were 2.8, 4.0, 0.35 and 0.82 mM respectively. Ammonium and structural analogues of glutamine and ammonium viz, methionine sulfone (MSO), methionine-DL-sulfoximine (MSX), ethylenediamine (EDA) and glyphosine significantly inactivated the enzyme activity while azaserine showed partial inhibition. Polyclonal antibodies raised against purified GS protein of N. muscorum showed high specificity to both crude and pure GS preparations in different immunological assays like double diffusion, rocket immunoelectrophoresis, ELISA and western blotting. With serological procedure a microquantity of GS-antigen upto 2 ng in vivo and immunological relationship of the protein with different strains have been documented.