An NADH-dependent glutamate synthase has been purified 500-fold from the plant cytoplasm fraction of Lupinus angustifolius nodules. It consists of a single polypeptide chain, M , 235000. The optimum pH is 8.5, at which K , values for 2-oxoglutarate, glutamine and NADH are 39 pM, 400 pM and 1.3 pM respectively. The catalytic centre activity is of the order of 70 s-' and is independent of pH between 6.5 and 9.5. Glutamate synthase is inhibited by glutamic acid, oxaloacetic acid, aspartic acid and asparagine, all competitive with 2-oxoglutarate; and by NAD' , which is competitive with NADH.There is evidence of two flavine prosthetic groups per enzyme molecule.Although the nitrogenase enzyme of nitrogen fixation has been extensively studied and is comparatively well understood, the enzymes that metabolise the ammonia produced by the nitrogenase reaction are relatively poorly understood.Robertson et al. [l] first showed the presence of a NADH-dependent glutamate synthase in the plant cytoplasm fraction of the nodule. The level of this enzyme was shown to increase dramatically in developing nodules with the onset of nitrogen fixation, suggesting a role in the overall ammonia assimilation system.Recently Scott et al.[2] suggested a pathway for nitrogen metabolism in the nodule, involving glutamate synthase and three other enzymes : glutamine synthetase, aspartate aminotransferase and asparagine synthetase.This laboratory is currently investigating ammonia assimilation by nitrogen-fixing lupin nodules. We now report the purification and some kinetic properties of the plant cytoplasmic NADH-dependent glutamate synthase. The reaction catalysed is:Abbreviation. Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulphonic acid. EXPERIMENTAL PROCEDURE MaterialsNADH (grade 111), bovine serum albumin (fraction V) and oxaloacetic acid were obtained from Sigma Chemical Co. Amino acids and other keto acids were from BDH Chemicals Ltd. All other reagents were analytical grade purity. PuriJj:cation of EnzymeLupins (Lupinus angustifolius L. Uniwhite) were grown as previously described [I]. Nodules (26.5 g) were harvested from 160 18-day-old plants, extracted and centrifuged to remove bacteroids [l]. The supernatant was then treated with 0.01 vol. of 0.1 M phenylmethylsulphonyl fluoride in isopropanol and centrifuged 20 min at 0 "C, 30000 x g to remove particulate material, giving an extract of the plant cytoplasm fraction. In the subsequent enzyme purification all steps were carried out at 0-4 "C. 0.1 M potassium phosphate buffer, pH 7.6, containing 0.5 % mercaptoethanol was used throughout, with modifications as described.Glutamate synthase was precipitated from the plant cytoplasm extract between 35 % and 50 % saturation with ammonium sulphate (209 g lP1, plus 94 g I-') and was resuspended in 2-3 ml of phosphate buffer to which had been added 1 mg disodium EDTA,