Catalyzes the reaction shown in Figure 1, which is equivalent to that catalyzed by the wellstudied enzyme glutathione reductase. bis–glutamylcystine (-Glu-Cys), which lacks the glycine moiety of glutathione, is usually a big intracellular thiol in halobacteria, Archaea which can be adapted for life in high-salt environments. Maintenance of reduced -Glu-Cys in halobacteria needs GCR. Here we report the identification from the gene encoding GCR in Halobacterium sp. NRC-1. The enzyme is mis-annotated as a mercuric reductase. GCR belongs towards the pyridine nucleotide disulfide reductase family members, and is found only in halobacteria. Nevertheless, some halobacteria lack GCR, suggesting that there is certainly diversity with respect to mechanisms for keeping the redox state from the cytoplasm and protection against oxidative damage even inside the Halobacterium clade.NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptEXPERIMENTAL PROCEDURESGrowth of Halobacterium sp. NRC-Halobacterium sp. NRC-1 and its genomic DNA were generous gifts from Dr. Nitin Baliga (Institute for Systems Biology, Seattle, WA). A single colony grown for a single week on Halobacterium halobium complex agar medium at 42 was inoculated into five mL of H. halobium complex medium (CM)ten in a 15 mL culture tube. Following 4 days of incubation at 42 with shaking at 250 rpm, the culture was added to one hundred mL of CM in a 500 mL Erlenmeyer flask and incubated for four much more days at 42 with shaking. At that point, ten mL aliquots on the culture had been applied to inoculate 1 L of H. halobium complex medium in every of seven four L Erlenmeyer flasks. Cultures had been incubated for four days and the cells were harvested by centrifugation at four,000 ?g at area temperature for 40 min. Cell pellets were stored at -80 prior to use.Chemical compounds and also other components Bis–glutamylcystine was prepared by passing O2 via an aqueous answer of -GluCys (94 mg dissolved in 3.0 mL of deionized water). The pH in the solution was adjusted to 8.0 with NH4OH prior to the oxidation reaction.11 The purity on the lyophilized bis-glutamylcystine was assessed by H1- and C13-NMR in D2O. The product was more than 99 pure and no remaining -Glu-Cys was detectable. 1H-NMR (400 MHz, D2O), 4.48 (dd, J = 4.0, 9.2 Hz, 1 H); 3.76 (dd, J = five.2, six.eight Hz, 1 H); 3.23 (dd, J = 4.0, 14 Hz, 1 H); two.93 (dd, J = 9.2, 14 Hz, 1 H); 2.47 (m, 2 H); and two.16 (m, 2H). 13C-NMR (75 MHz, D2O), 176.9, 174.3, 174.1, 54.3, 54.2, 39.8, 31.7 and 26.five. Butyl-Sepharose FF, HiTrap chelating HP, and HisTrap HP (immobilized Ni2+) resins have been purchased from GE Healthcare Biosciences (Pittsburgh, PA). Immobilized Cu2+ resin was ready from HiTrap chelating HP resin applying 0.1 M CuCl2 following the manufacturer’s instruction. GCR activity assay GCR activity was detected as described by Caspase Inhibitor Species Sundquist and Fahey.12 A single unit of enzyme activity is defined as the quantity of enzyme that catalyzes P2Y2 Receptor Compound conversion of 1 mol of substrateBiochemistry. Author manuscript; offered in PMC 2014 October 28.Kim and CopleyPageper minute with 1 mM bis–glutamylcystine and 0.42 mM NADPH. For reactions with varying concentrations of bis–glutamylcystine, the concentration of NADPH was held continuous at 1.7 mM. Mercuric reductase activity assay Mercuric reductase activity was assayed by following the oxidation of NADPH at 340 nm at room temperature.13 Assays were carried out in 50 mM sodium phosphate, pH six.7, containing 3 M KCl, 1.three M NaCl, 1 mM EDTA, 0.34 mM NADPH and as much as 1 mM HgCl2. Purification of GCR from Ha.