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., Ohman, L., Chapman, B.E., Jacobsson, U., Kuchel, P.W., Häggström, 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 1H/15N 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 1H/15N 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 [5-15N] glutamine was selectively incorporated into 2-oxoglutarate forming [2-15N] glutamate with a specific activity of 4.15 +/- 0.21 nmol [2-15N] 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.
Doverskog
M
M
Department of Biotechnology, Royal Institute of Technology, Stockholm, Sweden. magnusd@biotech.kth.se
Jacobsson
U
U
Chapman
B E
BE
Kuchel
P W
PW
Häggström
L
L
D000818:Animals; D002460:Cell Line; D002474:Cell-Free System; D005970:Glutamate Synthase / Q000032:analysis* / Q000378:metabolism; D005972:Glutaminase / Q000378:metabolism; D005973:Glutamine / Q000378:metabolism; D007656:Ketoglutaric Acids / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D009243:NAD / Q000494:pharmacology*; D009587:Nitrogen Isotopes; D000644:Quaternary Ammonium Compounds / Q000378:metabolism; D013053:Spectrophotometry; D018411:Spodoptera / Q000201:enzymology*
D007656:Ketoglutaric Acids; D009587:Nitrogen Isotopes; D000644:Quaternary Ammonium Compounds; D005973:Glutamine; D009243:NAD; D005970:Glutamate Synthase; D005972:Glutaminase