Determination of NADH-dependent glutamate synthase (GOGAT) in Spodoptera frugiperda (Sf9) insect cells by a selective 1H/15N NMR in vitro assay
Determination of NADH-dependent glutamate synthase (GOGAT) in Spodoptera frugiperda (Sf9) insect cells by a selective 1H/15N NMR in vitro assay
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DOI：
10.1016/s0168-1656(00)00215-7
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发表时间：
2000-04-14
影响因子：
4.1
通讯作者：
Häggström, L
中科院分区：
文献类型：
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作者：
Doverskog, M;Jacobsson, U;Häggström, L
这是两篇论文中的第二篇[Drews，M.，Doverskog，M.，Qhman，L.，Chapman，B.E.，Jacobson，U，Kuchel，P.W.，Haggstrom，L.，2000。草地贪夜蛾(Sf9)昆虫细胞中谷氨酰胺代谢途径：存在氮同化系统的证据，以及H-1/N-15核磁共振的代谢开关。J.Biotechnol。78、23-37]。其总体目标一直是确定和表征Sf9细胞中的谷氨酰胺代谢。用改进的H-1/N-15自旋回波和梯度增强多量子相干核磁共振技术研究了果蝇(S.rugiperda)昆虫细胞无细胞提取物中谷氨酸合成酶(GOGAT)活性的存在。从无血清培养的细胞中制备无细胞提取物。测定条件以常规的分光光度法和层析法为基础。核磁共振数据表明，在无细胞提取液中，来自[5-N-15]谷氨酰胺的氮被选择性地结合到2-氧戊二酸中，生成比活性为4.15+/-0.21nmoL[2-N-15]谷氨酸min(-1)(mg总蛋白)(-1)的[2-N-15]谷氨酸。该酶的活性完全依赖于NADH作为辅酶，并被1 mM氮杂丝氨酸完全抑制。结果表明，Sf9细胞具有NADH-GOGAT活性。此外，核磁共振方法的高度特异性使得能够区分谷氨酰胺酶和谷氨酸脱氢酶的竞争反应。(C)2000 Elsevier Science B.V.保留所有权利。
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.