PURIFICATION AND CHARACTERIZATION OF NADH-DEPENDENT GLUTAMATE SYNTHASE FROM THE SILKWORM FAT-BODY (BOMBYX-MORI)

NADH-dependent glutamate synthase (NADH-GOGAT; EC 1.4.1.14) was purifi ed 766-fold from the fat body of 5th instar larvae of the silkworm wit h a final specific activity of 13.8 units/mg protein by a procedure in cluding ammonium sulfate fraction, Q-Sepharose HP ion exchange column chromatography, Blue Sepharose FF affinity column chromatography and S uperdex 200HR gel filtration. The purified enzyme yielded a single ban d corresponding to a molecular mass of 195 kDa by SDS-polyacrylamide g el electrophoresis. Molecular mass of the native enzyme was estimated to be 190 kDa by Superdex 200HR gel filtration, suggesting that the en zyme is composed of a monomeric polypeptide. The enzyme showed an abso rption spectrum with maximum values at 272, 375 and 435 nm, suggesting the presence of a flavin prosthetic group in the enzyme. The N-termin al amino acid sequence showed a high similarity to those of other GOGA Ts from plants, yeast and bacteria, but no similarity to other known p roteins was detected. The enzyme exhibited a strong specificity to the electron donor and substrates; NADH as electron donor, 2-oxoglutarate as amino acceptor and glutamine as amino donor were essential for the catalytic activity. The optimum pH was around 7.5, at which K-m value s for 2-oxoglutarate, glutamine and NADH were 17, 220 and 5.7 mu M, re spectively. Azaserine, 6-diazo-5-oxo-norleucine and p-chloromercuriben zoic acid were strong inhibitors of the activity. These results show t hat NADH-GOGAT in the silkworm fat body strongly resembles other eukar yotic NADH-GOGATs, suggesting that it plays a significant role in ammo nia assimilation in the same manner as the other enzymes. (C) 1998 Els evier Science Ltd. All rights reserved.