PURIFICATION AND CHARACTERIZATION OF GLUTAMINE-SYNTHETASE FROM THE GREEN-ALGA MONORAPHIDIUM-BRAUNII

Glutamine synthetase (EC 6.3.1.2] has been purified from the green alg a Monoraphidium braunii. The enzyme was purified by a method which inc luded consecutive chromatographies on: DEAE Sepharose, Blue Sepharose, second DEAE Sepharose, Sephacryl S-300 and Phenyl Sepharose CL-4B. Th e apparent molecular weight of the GS subunit was approximately 42 000 . Since the undissociated enzyme has a molecular weight of 295 000, M. braunii GS can be considered as a plant type GS, probably with octame ric structure. Purified glutamine synthetase was inhibited by some ami no acids and nucleotides. The Stokes radius of the native enzyme was 6 .13 nm. Values for apparent Michaelis constants of the physiological a ctivity of the purified enzyme for glutamate, ATP, and ammonium were 5 .7, 0.85 and 0.05 mM respectively. Alanine, glycine, aspartate and ser ine inhibited both transferase and synthetic activities of GS. Glutami ne synthetase from M. braunii was inhibited by p-hydroxymercuribenzoat e, the effect being reversed by treatment with dithioerytritol. (C) 19 97 Elsevier Science Ireland Ltd.