A simple and sensitive method for glutamine:fructose-6-phosphate amidotransferase assay

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-phophate Na 2 (5 mM) were added to the buffer for scraping the cells. The range of protein content in the samples was 80–150 μg. 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.