16 mM NADH. The 1 mL reverse reaction assay (oxidative deamination) was prepared by adding 100 mM Phosphate buffer (pH 7.0); 100 mM L-glutamate; and 2 mM NAD+. The assay reactions were initiated by the addition of 10 μg M. smegmatis crude protein extract. The forward or aminating reactions were assayed by measuring the oxidation of NADPH or NADH spectrophotometrically at 340 nm. The reverse or deaminating reactions were assayed by measuring the reduction of NADP+ or NAD+ at 340 nm. Specific enzyme activities were calculated using the NAD(P)H extinction
co-efficient of 6.22 cm2/μmole. One unit of selleck screening library enzyme activity was defined as 1 nmole of coenzyme (NAD(P)H) oxidized or reduced per minute, per milligram protein added. A two-way ANOVA using a mixed model with the correct nested terms was used to analyse the data. Glutamine synthetase activity assay Total GS activity was assayed using the γ-glutamyl-transferase assay as described elsewhere [58]. Briefly, total GS activity was assayed in the presence of 0.3 mM Mn2+ as the activity of both adenylylated and de-adenylylated forms of GS are measured under these conditions. The reaction was initiated by the addition of 10 μg M. smegmatis crude protein extract and allowed to proceed for 30 min at 37°C. The reaction was halted
by the addition of a stop mix IWR 1 (1 M FeCl3.6H2O, 0.2 M Trichloroacetic acid and 7.1% v/v HCl) and the samples were briefly centrifuged in order to remove any precipitate that may have formed. The production of γ-glutamylhydroxamate was determined by measuring the absorbance at 540 nm. One unit of enzyme activity was defined as the amount of enzyme producing 1 μmole γ-glutamylhydroxamate/min/mg protein in the transfer
reaction. A technical replicate of each enzyme assay was measured and each experiment Etofibrate was repeated at least three times. A two-way ANOVA using a mixed model with the correct nested terms was used to analyse the data. RNA preparation M. smegmatis cells were collected by centrifugation (Eppendorf Centrifuge 5810R) and resuspended in 1 ml Trizol (Invitrogen). The cell suspension was ribolysed (Fastprep FP120, Bio101 Savant) in a 2.0 ml screw cap microtube (Quality Scientific Plastics) containing 0.5 mm glass beads at a maximum speed setting of 6.0 for 20 seconds. The tubes were immediately placed on ice for 1 minute to dissipate the heat caused by friction during the ribolyzing process. This homogenisation step was repeated 3-4 times and the cooled homogenate was TPCA-1 datasheet incubated at room temperature for 5 minutes to allow dissociation of nucleoprotein complexes. A total of 250 μl chloroform was added to the mixture which was rapidly inverted for the first 20 seconds, and then periodically thereafter for a further 5 minutes at room temperature. The samples were centrifuged at 18630 × g (4°C) for 10 min and the aqueous phase removed.