N and 200 mM DMSO.Western blot analysisProteins were transferred to nitrocellulose membrane (Whatman) after PAGE, and western blots were performed using anti-polyhistidine peroxidase conjugate antibodies (Sigma) and Super Signal WestPico Chemiluminescent Substrate (Interchim), according to the manufacturer’s instructions. Luminescence was detected in a G-Box (Syngene).Enzymatic activityYedY reductase activity was spectrophotometrically assayed at 600 nm using reduced benzyl viologen as the electron donor (600 = 10.4 mM-1.cm-1) and DMSO as the substrate, in a glovebox workstation (MBRAUN Labstar) flushed with nitrogen. Each reaction mixture (1 ml) contained 100 mM MES (pH 6.0), 0.2 mM benzyl viologen reduced with sodium dithionite, and a variable S28463 web concentration of DMSO. The initial reaction rates were plotted as a function of DMSO concentration, and the nonlinear regression of the Michaelis enten equation was calculated PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/26024392 with SigmaPlot analysis software. To MK-886 supplier determine kinetic parameters, three experiments with two to three independent biological samples were used.EPR spectroscopyX-band EPR spectra were collected at 9.4 GHz using a Bruker ELEXSYS 500E spectrometer fitted with an Oxford Instruments ESR 900 helium flow cryostat. Simulations of the EPR spectra were performed using the Matlab toolbox EasySpin [40].Sabaty et al. BMC Biochemistry 2013, 14:28 http://www.biomedcentral.com/1471-2091/14/Page 12 of7.8.9. 10. 11. 12.13.14.15. 16.17.18.19.20.21.22. 23.24. 25.26.27.28.29.sulfite oxidase homologue from Escherichia coli. Inorg Chem 2011, 50:741?48. Dreyfus C, Pignol D, Arnoux P: Expression, purification, crystallization and preliminary X-ray analysis of an archaeal protein homologous to plant nicotianamine synthase. Acta Crystallogr Sect F: Struct Biol Cryst Commun 2008, 64:933?35. Beckham KS, Byron O, Roe AJ, Gabrielsen M: The structure of an orthorhombic crystal form of a `forced reduced’ thiol peroxidase reveals lattice formation aided by the presence of the affinity tag. Acta Crystallogr Sect F: Struct Biol Cryst Commun 2012, 68:522?26. Pugsley AP: The complete general secretory pathway in gram-negative bacteria. Microbiol Rev 1993, 57:50?08. Danese PN, Silhavy TJ: Targeting and assembly of periplasmic and outermembrane proteins in Escherichia coli. Annu Rev Genet 1998, 32:59?4. Berks BC, Sargent F, Palmer T: The Tat protein export pathway. Mol Microbiol 2000, 35:260?74. Santini CL, Ize B, Chanal A, Muller M, Giordano G, Wu LF: A novel secindependent periplasmic protein translocation pathway in Escherichia coli. Embo J 1998, 17:101?12. Hatzixanthis K, Clarke TA, Oubrie A, Richardson DJ, Turner RJ, Sargent F: Signal peptide-chaperone interactions on the twin-arginine protein transport pathway. Proc Natl Acad Sci U S A 2005, 102:8460?465. Genest O, Neumann M, Seduk F, Stocklein W, Mejean V, Leimkuhler S, Iobbi-Nivol C: Dedicated metallochaperone connects apoenzyme and molybdenum cofactor biosynthesis components. J Biol Chem 2008, 283:21433?1440. Genest O, Mejean V, Iobbi-Nivol C: Multiple roles of TorD-like chaperones in the biogenesis of molybdoenzymes. FEMS Microbiol Lett 2009, 297:1?. Dow JM, Gabel F, Sargent F, Palmer T: Characterisation of a pre-export enzyme-chaperone complex on the twin-arginine transport pathway. Biochem J 2013, 452. Sambasivarao D, Turner RJ, Simala-Grant JL, Shaw G, Hu J, Weiner JH: Multiple roles for the twin arginine leader sequence of dimethyl sulfoxide reductase of Escherichia coli. J Biol Chem 2000, 275:225.N and 200 mM DMSO.Western blot analysisProteins were transferred to nitrocellulose membrane (Whatman) after PAGE, and western blots were performed using anti-polyhistidine peroxidase conjugate antibodies (Sigma) and Super Signal WestPico Chemiluminescent Substrate (Interchim), according to the manufacturer’s instructions. Luminescence was detected in a G-Box (Syngene).Enzymatic activityYedY reductase activity was spectrophotometrically assayed at 600 nm using reduced benzyl viologen as the electron donor (600 = 10.4 mM-1.cm-1) and DMSO as the substrate, in a glovebox workstation (MBRAUN Labstar) flushed with nitrogen. Each reaction mixture (1 ml) contained 100 mM MES (pH 6.0), 0.2 mM benzyl viologen reduced with sodium dithionite, and a variable concentration of DMSO. The initial reaction rates were plotted as a function of DMSO concentration, and the nonlinear regression of the Michaelis enten equation was calculated PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/26024392 with SigmaPlot analysis software. To determine kinetic parameters, three experiments with two to three independent biological samples were used.EPR spectroscopyX-band EPR spectra were collected at 9.4 GHz using a Bruker ELEXSYS 500E spectrometer fitted with an Oxford Instruments ESR 900 helium flow cryostat. Simulations of the EPR spectra were performed using the Matlab toolbox EasySpin [40].Sabaty et al. BMC Biochemistry 2013, 14:28 http://www.biomedcentral.com/1471-2091/14/Page 12 of7.8.9. 10. 11. 12.13.14.15. 16.17.18.19.20.21.22. 23.24. 25.26.27.28.29.sulfite oxidase homologue from Escherichia coli. Inorg Chem 2011, 50:741?48. Dreyfus C, Pignol D, Arnoux P: Expression, purification, crystallization and preliminary X-ray analysis of an archaeal protein homologous to plant nicotianamine synthase. Acta Crystallogr Sect F: Struct Biol Cryst Commun 2008, 64:933?35. Beckham KS, Byron O, Roe AJ, Gabrielsen M: The structure of an orthorhombic crystal form of a `forced reduced’ thiol peroxidase reveals lattice formation aided by the presence of the affinity tag. Acta Crystallogr Sect F: Struct Biol Cryst Commun 2012, 68:522?26. Pugsley AP: The complete general secretory pathway in gram-negative bacteria. Microbiol Rev 1993, 57:50?08. Danese PN, Silhavy TJ: Targeting and assembly of periplasmic and outermembrane proteins in Escherichia coli. Annu Rev Genet 1998, 32:59?4. Berks BC, Sargent F, Palmer T: The Tat protein export pathway. Mol Microbiol 2000, 35:260?74. Santini CL, Ize B, Chanal A, Muller M, Giordano G, Wu LF: A novel secindependent periplasmic protein translocation pathway in Escherichia coli. Embo J 1998, 17:101?12. Hatzixanthis K, Clarke TA, Oubrie A, Richardson DJ, Turner RJ, Sargent F: Signal peptide-chaperone interactions on the twin-arginine protein transport pathway. Proc Natl Acad Sci U S A 2005, 102:8460?465. Genest O, Neumann M, Seduk F, Stocklein W, Mejean V, Leimkuhler S, Iobbi-Nivol C: Dedicated metallochaperone connects apoenzyme and molybdenum cofactor biosynthesis components. J Biol Chem 2008, 283:21433?1440. Genest O, Mejean V, Iobbi-Nivol C: Multiple roles of TorD-like chaperones in the biogenesis of molybdoenzymes. FEMS Microbiol Lett 2009, 297:1?. Dow JM, Gabel F, Sargent F, Palmer T: Characterisation of a pre-export enzyme-chaperone complex on the twin-arginine transport pathway. Biochem J 2013, 452. Sambasivarao D, Turner RJ, Simala-Grant JL, Shaw G, Hu J, Weiner JH: Multiple roles for the twin arginine leader sequence of dimethyl sulfoxide reductase of Escherichia coli. J Biol Chem 2000, 275:225.
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