Isolation and characterization of NADH-glutamate synthase from pea (Pisum sativum L.)

Abstract

Both ferredoxin-glutamate synthase (EC 1.4.7.1) and NADH-glutamate synthase (EC 1.4.1.14) were isolated separately on DEAE-cellulose chromatography from etiolated pea shoots. The latter enzyme was purified 1,400-fold by ammonium sulfate fractionation and column chromatographies of DEAE-cellulose, Sephadex G-200 and blue-Sepharose. The enzyme had a molecular weight of 220,000 and an isoelectric point of 4.3. The optimum pH was 7.6. Apparent Km values for l-glutamine, 2-oxoglutarate and NADH were 400, 37 and 4 µm, respectively. The enzyme had its absorption maxima at 275, 375 and 440 nm, suggesting that pea NADH-glutamate synthase is a flavoprotein. It showed NADH-diaphorase activity toward ferricyanide and 2,6-dichlorophenol indophenol as the electron acceptor. Sulfhydryl reagents, metal-chelating reagents, phthalein acids and azaserine were strong inhibitors. Ammonium and phosphate ions enhanced the enzyme activity.