| Literature DB >> 15165751 |
Fei Ye1, Hiroshi Maegawa, Katsutaro Morino, Atsunori Kashiwagi, Ryuichi Kikkawa, Mingzhi Xie, Zhufang Shen.
Abstract
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.Entities:
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Year: 2004 PMID： 15165751 DOI： 10.1016/j.jbbm.2003.02.001
Source DB: PubMed Journal: J Biochem Biophys Methods ISSN： 0165-022X