Glutamine glutamate glotm assay kit

The activity and inhibition profiles of purified MBP-fused PfGS-I or human GS-II (hGS-II) (BioVision Inc., Milpitas, CA, USA) in the presence or absence of AST were estimated from glutamine production using Glutamine/Glutamate-GLO^TM Assay Kit (Promega, Madison, WI, USA). The assay mixture (50 mM imidazole-HCl buffer (pH 7.0), 7.6 mM ATP, 50 mM magnesium chloride, 50 mM ammonium chloride) with or without the indicated concentration of GS enzyme was preincubated in the presence or absence of the indicated concentrations of AST for 15 min at room temperature. The reaction was initiated through the addition of L-glutamate at 0.1 mM, the final concentration. The reaction mixtures were incubated at 37 °C with shaking at 300 rpm for the indicated time. The reactions were terminated by chilling the reaction mixture in ice water and filtered with 30 kDa Amicon Ultra centrifugal filters at 4 °C. Glutamate concentrations in the filtered samples were determined using a Glutamine/Glutamate-GLO^TM Assay as instructed in the kit’s manual, from which glutamine concentrations were estimated by subtracting the glutamate concentrations from the initial concentrations (0.1 mM). The EC₅₀ (half maximal effective concentration) of AST for PfGS-I was calculated using Sigma Plot (Systat Software, Inc., Sun Jose, CA, USA).

Pyruvate assay kit (Abnova), succinate colorimetric assay kit (BioVision), and acetate colorimetric assay kit (Sigma) were used for metabolite quantification. Procedures were followed according to the manuals. M. bovis BCG was grown in 6% glycerol Sauton’s medium for 9 or 11 days before culture filtrates were harvested for extracellular metabolite quantification. In some experiments, cells were pelleted and lysed by sonication prior to intracellular metabolite quantification. The concentrations of these metabolites were calculated based on their corresponding standard curves and normalized by protein concentrations.
Glutamate/glutamine was measured using the glutamine/ glutamate-Glo^TM assay kit (Promega) according to the manual. The glutamate/glutamine level [measured as relative light unit (RLU)] was recorded by Lumat LB 9507. Data were normalized by OD600.