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

This is the second of two papers [Drews, M., Doverskog, M., Qhman, L., Chapman, B.E., Jacobsson, U., Kuchel, P.W., Haggstrom, L., 2000. Pathways of glutamine metabolism in Spodoptera frugiperda (Sf9) insect cells: evidence for 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 presence of glutamate synthase (GOGAT) activity was investigated in cell-free extracts of S. frugiperda (Sf9) insect cells by modified H-1/N-15 spin-echo and gradient enhanced multiple quantum coherence NMR spectroscopy techniques. 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] glutamine was selectively incorporated into 2-oxoglutarate forming [2-N-15] glutamate 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 activity was exclusively dependent on NADH as coenzyme and was completely inhibited by 1 mM azaserine. From the results obtained, we conclude that Sf9 cells possess NADH-GOGAT activity. Furthermore, the high specificity of the NMR method enables distinction of competing reactions from glutaminase and glutamate dehydrogenase. (C) 2000 Elsevier Science B.V. All rights reserved.