Determination of NADH-dependent glutamate synthase (GOGAT) in Spodoptera frugiperda (Sf9) insect cells by a selective H-1/N-15 NMR in vitro assay
2000 (English)In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 79, no 1, 87-97 p.Article in journal (Refereed) Published
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.
Place, publisher, year, edition, pages
2000. Vol. 79, no 1, 87-97 p.
Spodoptera frugiperda (Sf9) insect cells, glutamate synthase, GOGAT, glutamine metabolism, H-1/N-15 nuclear magnetic resonance (NMR), bombyx-mori, ammonia assimilation, aspergillus-nidulans, agaricus-bisporus, amino-acids, nitrogen, metabolism, silkworm, dehydrogenase, spectroscopy
IdentifiersURN: urn:nbn:se:kth:diva-19723ISI: 000086786600008OAI: oai:DiVA.org:kth-19723DiVA: diva2:338415
QC 201005252010-08-102010-08-10Bibliographically approved