O. esters (POE) suggest that POE may undergo ready hydrolysis to POA, accounting for their activity against is the only mycobacterial species that is susceptible to PZA and POA. However, in culture, the antimycobacterial activity of both these compounds is usually observed only in an acidic medium. It has been shown that acidic pH allows for the intracellular accumulation of POA (30), yet the reason for acidic-pH-dependent POA accumulation remains elusive. (and probably other mycobacterial species), lacks an efficient POA efflux mechanism, which allows POA accumulation. This deficient POA efflux mechanism has been suggested as the origin of the unique susceptibility of to PZA among mycobacteria (30). Nonetheless, since hydrolytically stable POE such as genes from BCG, and confer resistance to 5-Cl-PZA. The observations that 5-Cl-PZA inhibits fatty acid synthase I (FASI) in and that both 5-Cl PZA and PZA inhibit FASI (albeit with the proviso that PZA requires acidic conditions) (32) suggest that FASI is usually a potential antituberculosis drug target. The inhibition of FASI by 5-Cl-PZA has been verified independently, but the inhibition of FASI by PZA was not confirmed (4). FAS is essential for cell survival. FAS (E.C. 220.127.116.11) catalyzes the sequential condensation of acetyl-coenzyme A (CoA) and malonyl-CoA to form long chain fatty acids (6, 17). The structure of this enzyme complex differs dramatically in prokaryotes and eukaryotes (14). In most prokaryotes, the synthases are typically composed of at least seven peptides that represent the individual enzyme components and are generally classified as type II synthases (18, 23). However in mammals and mycobacteria the synthase activity is usually carried out by single high-molecular-weight, multifunctional peptide chains or type I synthases (6, 20, 26). Since fatty acid synthesis in bacteria is essential for cell survival, the enzymes involved in this pathway have emerged as promising targets for antimicrobial brokers (11, 12). As mentioned earlier, FASI was inhibited by 5-Cl-PZA (4, 32). Since Capsaicin eukaryotic microbial cells dependent upon endogenously synthesized fatty acids will express type I FAS, inhibitors of type I FAS could reasonably be anticipated to be inhibitors of microbial cell growth (12). To pursue the study of FASI as a drug target, a cell-free NADPH oxidation assay of FASI activity was employed, where FASI was isolated from a recombinant strain of (strain, (gene, was Capsaicin cultivated in 7H9 medium supplemented with NaCl (0.85 g/liter), glucose (0.2%), glycerol (0.2%), and Tween 80 (0.05%). Isolation and purification of FASI. Purification of FASI from mc22700 was effected using a slight modification of the procedures reported by Solid wood et al. (25) and Boshoff et al. (4). mc22700 was produced to an optical density at 600 nm of 1 1 to 1 1.3 at 37C. Cells Capsaicin were pelleted, washed with phosphate-buffered saline, and frozen at ?78C until use. All subsequent isolation and purification actions were done at 4C. Cells (11 g) were lysed using a bead beater in 0.1 M potassium phosphate buffer (3-ml/g pellet, pH 7.2; 11 mM dithiothreitol [DTT], 1 mM EDTA, 2 mM phenylmethylsulfonyl fluoride). The lysed cells were centrifuged at 6,000 for 30 min, and the supernatant was treated with streptomycin sulfate (0.3 mg/ml) and recentrifuged at 18,000 for 45 min. The supernatant was then further ultracentrifuged at 40,000 rpm for 90 min. Solid ammonium sulfate was then added slowly to the supernatant to 1 1.4 M. The mixture was stirred for 30 min and then centrifuged (18,000 for 30 min). IL17RA To induce the precipitation of FASI, the resulting supernatant was then brought to 2.0 M in ammonium sulfate, stirred for 30 min, and centrifuged. The pellet from the 2 2.0 M ammonium sulfate precipitation was suspended in 0.15 M potassium phosphate buffer (10 ml, pH 7.2; 1 mM DTT, 1 mM EDTA).
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