- Characterization of Deinococcus radiophilus Thioredoxin Reductase Active with Both NADH and NADPH
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Hee-Jeong Seo , Young Nam Lee
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J. Microbiol. 2010;48(5):637-643. Published online November 3, 2010
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DOI: https://doi.org/10.1007/s12275-010-0283-7
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Thioredoxin reductase (TrxR, EC 1.6.4.5) of Deinococcus radiophilus was purified by steps of sonication, ammonium sulfate fractionation, 2'5' ADP Sepharose 4B affinity chromatography, and Sephadex G-100 gel filtration. The purified TrxR, which was active with both NADPH and NADH, gave a 368 U/mg protein of specific activity with 478-fold purification and 18% recovery from the cell-free extract. An isoelectric point of the purified enzymes was ca. 4.5. The molecular weights of the purified TrxR estimated by PAGE and gel filtration were about 63.1 and 72.2 kDa, respectively. The molecular mass of a TrxR subunit is 37 kDa. This suggests that TrxR definitely belongs to low molecular weight TrxR (L-TrxR). The Km and Vmax of TrxR for NADPH are 12.5 μM and 25 μM/min, whereas those for NADH are 30.2 μM and 192 μ M/min. The Km and Vmax for 5, 5'-dithio-bis-2-nitrobenzoic acid (DTNB, a substituted substrate for thioredoxin) are 463 μM and 756 μM/min, respectively. The presence of FAD in TrxR was confirmed with the absorbance peaks at 385 and 460 nm. The purified TrxR was quite stable from pH 3 to 9, and was thermo-stable up to 70°C. TrxR activity was drastically reduced (ca. 70%) by Cu2+, Zn2+, Hg2+, and Cd2+, but moderately reduced (ca. 50%) by Ag+. A significant inhibition of TrxR by N ethylmaleimide suggests an occurrence of cysteine at its active sites. Amino acid sequences at the N-terminus of purified TrxR are H2N-Ser-Glu-Gln-Ala-Gln-Met-Tyr-Asp-Val-Ile-Ile-Val-Gly-Gly-Gly-Pro-Ala-Gly-Leu-Thr-Ala-COOH. These sequences show high similarity with TrxRs reported in Archaea, such as Methanosarcina mazei, Archaeoglobus fulgidus etc.
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Citations
Citations to this article as recorded by  - Purification of thioredoxin reductase from Spirulina platensis by affinity chromatography and investigation of kinetic properties
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