一种用于谷氨酰胺：果糖-6-磷酸酰胺转移酶测定的简单灵敏方法。
A simple and sensitive method for glutamine:fructose-6-phosphate amidotransferase assay.
作者信息
Ye Fei, Maegawa Hiroshi, Morino Katsutaro, Kashiwagi Atsunori, Kikkawa Ryuichi, Xie Mingzhi, Shen Zhufang
机构信息
Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China.
出版信息
J Biochem Biophys Methods. 2004 Jun 30;59(3):201-8. doi: 10.1016/j.jbbm.2003.02.001.
For measuring glutamine:fructose-6-phosphate amidotransferase (GFAT) activity in cultured cells, an enzyme method -GDH method- was set up with high-efficiency, high-sensitivity and simple operation by determining the formed glutamate. During the process of making samples, reduced glutathione (GSH, 5 mM) and glucose-6-phosphate Na2 (5 mM) were added to the buffer for scraping the cells. The range of protein content in the samples was 80-150 microg. In the GFAT activity assay, the end product reduced acetylpyridine adenine dinucleotide (APADH) was determined at 370 nm directly. The suitable concentrations of the reactants fructose-6-phosphate (F-6-P), glutamine, acetylpyridine adenine dinucleotide (APAD) and glutamate dehydrogenase (GDH) were 0.8, 6 and 0.3 mM and 6 U, respectively. However, the excess of APAD may interfere with the APADH measurement. The reaction time course was 90 min. The GFAT activity in 3T3-L1, L6, HepG2 and HIRc cells were 1.84-8.51 nmol glutamate/mg protein.min.
为了测定培养细胞中谷氨酰胺
果糖-6-磷酸酰胺转移酶（GFAT）的活性，通过测定生成的谷氨酸建立了一种高效、高灵敏度且操作简单的酶法——GDH法。在制备样品的过程中，向用于刮取细胞的缓冲液中添加了还原型谷胱甘肽（GSH，5 mM）和葡萄糖-6-磷酸二钠（5 mM）。样品中的蛋白质含量范围为80 - 150微克。在GFAT活性测定中，直接在370 nm处测定终产物还原型乙酰吡啶腺嘌呤二核苷酸（APADH）。反应物果糖-6-磷酸（F-6-P）、谷氨酰胺、乙酰吡啶腺嘌呤二核苷酸（APAD）和谷氨酸脱氢酶（GDH）的合适浓度分别为0.8、6和0.3 mM以及6 U。然而，过量的APAD可能会干扰APADH的测量。反应时间进程为90分钟。3T3-L1、L6、HepG2和HIRc细胞中的GFAT活性为1.84 - 8.51纳摩尔谷氨酸/毫克蛋白质·分钟。
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