Determination of NADH-dependent glutamate synthase (GOGAT) in Spodoptera frugiperda (Sf9) insect cells by a selective H-1/N-15 NMR in vitro assay

This is the second of two papers [Drews, M., Doverskog, M., Qhman, L., Chap man, B.E., Jacobsson, U., Kuchel, P.W., Haggstrom, L., 2000. Pathways of gl utamine metabolism in Spodoptera frugiperda (Sf9) insect cells: evidence fo r the presence of the nitrogen assimilation system, and a metabolic switch by H-1/N-15 NMR. J. Biotechnol. 78, 23-37]. where the general goal has been to determine and characterise the glutamine metabolism in Sf9 cells. The p resence of glutamate synthase (GOGAT) activity was investigated in cell-fre e extracts of S. frugiperda (Sf9) insect cells by modified H-1/N-15 spin-ec ho and gradient enhanced multiple quantum coherence NMR spectroscopy techni ques. Cell-free extracts were prepared from cells cultured in a serum-free medium. The assay conditions were based on conventional spectrophotometric and chromatographic methods. NMR data showed that nitrogen from [5-N-15] gl utamine was selectively incorporated into 2-oxoglutarate forming [2-N-15] g lutamate with a specific activity of 4.15 +/- 0.21 nmol [2-N-15] glutamate min (-1) (mg total protein)(-1) in the cell-free extracts. The enzyme activ ity was exclusively dependent on NADH as coenzyme and was completely inhibi ted by 1 mM azaserine. From the results obtained, we conclude that Sf9 cell s possess NADH-GOGAT activity. Furthermore, the high specificity of the NMR method enables distinction of competing reactions from glutaminase and glu tamate dehydrogenase. (C) 2000 Elsevier Science B.V. All rights reserved.