Author Manuscript NIH-PA Author ManuscriptDetection of pyruvate in culture supernatants To quantify pyruvate, p-dimethylbenzaldehyde was utilised as a derivatizing agent because it does not react with the other ketoacids present (Holtzclaw and Chapman, 1977). Strains to become tested were grown overnight in 1 ml of wealthy medium by continuous shaking at 37 , washed with one hundred mM NaCl and inoculated (1:8) into minimal media with indicated supplements. Aliquots were taken periodically and optical density at 650 nm was recorded. The cells have been removed by centrifugation (1 min at 14.8 K g) along with the supernatants had been frozen at -80 for additional analysis. To determine the pyruvate concentration in the supernatant the following were added to one hundred l of sample: 375 l of 5 N KOH and 375 l of pdimethylaminobenzaldehyde resolution (four.9 mg ml-1 methanol). The mixture was allowed to react for 30 min at 37 immediately after which the absorbance was taken at 420 nm. The concentration of pyruvate inside the supernatants was determined utilizing a standard curve with several concentrations of sodium pyruvate. To make sure no interfering compounds have been becoming detected within the assay above, an aliquot of supernatant was depleted of pyruvate applying lactate dehydrogenase to reduce pyruvate to lactate utilizing NADH.D-Pantothenic acid To deplete pyruvate in one hundred l of supernatant, five units of lactate dehydrogenase and 1 mol NADH were added and permitted to react for 1 h.Plerixafor Subsequent analysis showed no absorbance corresponding to interfering compounds.PMID:23829314 Determination of total coenzyme A in cells Total coenzyme A levels have been determined making use of a previously described method (Allred and Guy, 1969). Briefly, strains to become tested were grown overnight in wealthy media, washed with 100 mM NaCl and inoculated (1:50) into minimal media. Cultures were grown to 0.four OD650, harvested by centrifugation (8000 g for 12 min), and frozen at -80 for future evaluation. Cells were resuspended in phosphate-buffered saline and disrupted by the addition of formic acid to 0.25 N and incubation on ice for 30 min, vortexing periodically. Cell debris was then separated from lysate by centrifugation (14.8 K g) for ten min. The lysate was then neutralized by the addition of NH4OH. Aliquots of lysate had been treated with dithiothreitol (0.7 final) to facilitate reductive cleavage of CoA thioesters. Quantification of CoA was performed by coupled enzymatic assay, the reactions contained the following per ml: 330 l of DTT-treated lysate, 250 mol Tris (pH 7.two), 50 mol KCl, 15 mol malate, 6 mol acetylphosphate, 1 mol NAD+, three.three U citrate synthase, 15 U malate dehydrogenase and 7.5 U phosphotransacetylase. The rate of NADH formation was determined by monitoring absorbance at 340 nm. Serine transhydroxymethylase activity For activity determination in crude extract, strains have been grown in rich media overnight, cells were pelleted and resuspended in NaCl. A culture (1:50 inoculum) was grown in minimal medium to 0.4 OD650, cells were harvested by centrifugation (8000 g for 12 min and frozen at -80 for future analysis. Cell pellets have been resuspended in 100 mM potassium phosphate buffer (pH 7.3) with 1 mM EDTA and disrupted by sonication. Cell debris was removed byMol Microbiol. Author manuscript; available in PMC 2014 August 01.Flynn et al.Pagecentrifugation (14.8 K g) for ten min. Activity was assayed by modifying a described protocol (Schirch et al., 1985). Each 1 ml assay integrated: 30 l clarified cell lysate (or 1.five g of purified protein), one hundred mol potassium phosphate (pH 7.two), 0.four mol.
