Abstract
Glutamine synthetase (GS), which catalyzes the conversion of glutamate and ammonia to glutamine, is widely distributed in animal tissues and cell culture lines. The importance of this enzyme is suggested by the fact that glutamine, the product of GS-catalyzed de novo synthesis reaction, is the most abundant free amino acid in blood (Smith and Wilmore, 1990). Glutamine is involved in many biological processes including serving as the nitrogen donor for biosynthesis, as an exchanger for the import of essential amino acids, as a means to detoxifying intracellular ammonia and glutamate, and as a bioenergetics nutrient to fuel the tricarboxylic acid (TCA) cycle (Bott et al., 2015). The method for the assay of GS enzymatic activity relies on its γ-glutamyl transferase reaction by measuring γ-glutamylhydroxamate synthesized from glutamine and hydroxylamine, and the chromatographic separation of the reaction product from the reactants (Deuel et al., 1978). An overview of the GS glutamyl transferase reaction can be found in Figure 1. GS activity was measured by a spectrophotometric assay at a specific wavelength of 560 nm using a microplate reader. The method is simple, and has a comparable sensitivity with those methods applying radioactively labelled substrates. This modified procedure has been applied to assay/determine GS activity in cultured cell lines including the human mammary epithelial MCF10A cells and the murine pre-B FL5.12 cells, and could be used to measure GS activity in other cell lines.Figure 1. An overview of the GS glutamyl transferase reaction
Keywords: Glutamine synthetase, Activity assay, Glutamate ammonia ligase, GLUL, GS
Materials and Reagents
Equipment
Software
Procedure
Representative data
The GS activity was reported as nmol min-1 g protein-1. The GS activity was expressed by the formula: γ-glutamylhydroxamate concentration (mM) x 100 μl/incubation time (min)/protein amount (g). For example, the concentration of γ-glutamylhydroxamate calculated from MCF10A vector cells was 2.378 mM as compared with standard curve. The added protein amount was 10.36 μg, and the incubation time at 37 °C was 120 min. The GS activity would be 2.378 x 100/120/10.36 x10-6 = 1.91 x 105 nmol min-1 g protein-1 as indicated (Figure 3). Figure 3. Myc induces GS activity. c-Myc was stably expressed in MCF10A cells. GS activity was determined and is shown as the mean plus SEM of at least five independent experiments. *P < 0.05. Student’s t-test was used to determine the P value (Bott et al., 2015).
Notes
The various incubation time (2-6 h) at 37 °C was determined by the amount of active GS expressed in different cell lines. Higher active GS expressed leads to shorter incubation time to reach the optimal amount of γ-glutamylhydroxamate. The optimal incubation time used in the assay may be adjusted for different cell lines.
Recipes
Acknowledgments
This protocol was adapted and modified from a previous study (Deuel et al., 1978). It was described in (Bott et al., 2015). This work was supported by grants from NIH (R01CA129536 and R01GM97355 to WXZ).
References
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