A simple and sensitive method for glutamine:fructose-6-phosphate amidotransferase assay.
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.
Ye
Fei
F
Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China. yefie@imm.ac.cn
Maegawa
Hiroshi
H
Morino
Katsutaro
K
Kashiwagi
Atsunori
A
Kikkawa
Ryuichi
R
Xie
Mingzhi
M
Shen
Zhufang
Z
D041721:3T3-L1 Cells / Q000201:enzymology*; D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005969:Glutamate Dehydrogenase / Q000032:analysis / Q000737:chemistry; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing) / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D022781:Hepatocytes / Q000201:enzymology*; D006801:Humans; D051379:Mice; D032446:Myoblasts / Q000201:enzymology*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013057:Spectrum Analysis / Q000379:methods*
D005969:Glutamate Dehydrogenase; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing)