20240305 Gray Figure 8 data

FIG 8 Glutathione reductase is inhibited by nickel but not by HOSCN. Glutathione and thioredoxin reductase activity was measured in cell-free lysates of E. coli MG1655. Glutathione reductase assays (A-C) contained 50 mM HEPES-KOH buffer (pH 8), 1.2 mM oxidized glutathione, 600 µM DTNB, 350 µM NADPH, and thioredoxin reductase assays (D) contained 50 mM HEPES-KOH buffer (pH 8), 500 nM E. coli thioredoxin 1 (TrxA), 0.1 mg ml-1 bovine serum albumin, 500 µM DTNB, and 240 µM NADPH. Reactions also contained the indicated concentrations of NiSO4, polyP, and/or EDTA, and were started by addition of cell lysate (1.63-16.3 µg total protein). DTNB oxidation to TNB was measured over time, using the TNB extinction coefficient at 412 nm of 14100 M-1 cm-1, then specific activities were calculated as nmol GSH or TrxA reduced min-1 mg-1 total protein (n=3-6 experimental replicates; error bars of 1 standard deviation). HOSCN-oxidized lysates (used as indicated in B and D) were prepared by incubating cell lysate (3.26 mg ml-1 total protein) for 5 min at room temperature with 3 mM HOSCN, then quenching unreacted HOSCN with an equal volume of 2 mM TNB. Asterisks indicate activities significantly different from that of (A, B, and D) lysate with no NiSO4 added or (C) lysate with 1 mM NiSO4 added (filled circles, one-way ANOVA with Holm-Sidak’s multiple comparisons test or mixed model with Dunnett’s multiple comparisons test)(* = P<0.05, ** = P<0.01, *** = P<0.001).