Partial purification and characterization of NADH-glutamate synthase from faba bean ( Vicia faba) root nodules

Abstract

The NADH-dependent glutamate synthase (EC 1.4.1.14) from the plant fraction of N 2-fixing faba bean ( Vicia faba) nodules has been purified 74-fold to a specific activity of about 3 μmol min −1 mg protein −1 with a final yield of 32%. The NADH-GOGAT activity was associated with a single form of the enzyme that behaved as a monomeric protein with a subunit molecular weight of 195 kDa and a native molecular weight from 222 to 236 kDa estimated by gel filtration or PAGE, respectively. The NADH-GOGAT band on SDS-PAGE was cut out and used to produce antibodies. Western blots of SDS-PAGE of crude nodule proteins revealed a 195 kDa polypeptide in root extracts but not in those of leaves or bacteroids. The antiserum also cross-reacted with a polypeptide of camparable molecular weight (195 kDa) from both amide and ureide transporting species legume nodules, indicating that some antigenic epitopes have been conserved between nodule NADH-GOGAT of different species.