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Ye, F. et al.

Ye, Maegawa, Morino, Kashiwagi, Kikkawa, Xie, Shen,

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.[1]

References

A simple and sensitive method for glutamine:fructose-6-phosphate amidotransferase assay. Ye, F., Maegawa, H., Morino, K., Kashiwagi, A., Kikkawa, R., Xie, M., Shen, Z. J. Biochem. Biophys. Methods (2004) [Pubmed]

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