Gssg reductase

Aortic sections were fixed with 4% PFA and blocked free thiol groups with 40 mM N-ethylmaleimide in buffer containing 25 mM HEPESs pH 7.4, 0.1 mM EDTA pH 8.0, and 0.01 mM Neocuproine with 1% Triton (v/v). After three times washes with PBS, mixed disulfides were deglutathionylated with 27 μg/ml E. coli GRX1, 4 U/ml GSSG reductase (Roche), 1 mM GSH, 1 mM NADPH and 1 mM EDTA in Tris pH 8.0 for 20 min at RT. Aortic tissues were washed three times with PBS and newly reduced thiol groups were labeled with 1 mM N-(3-maleimidylpropionyl) biocytin (MBP, Sigma Aldrich) for 1 h at RT. Tissues were incubated with 10 ug/ml streptavidin-conjugated FITC (Thermo Fisher Scientific) for 1 h at RT and nuclei counterstain with DAPI (Invitrogen). The images were captured with a fluorescent microscope (Leica) and a high-resolution digital camera (Olympus).

Pr-SSG was detected in situ in paraffin-embedded lung tissue as previously described^{76 (link)}. After dewaxing tissue samples in three changes of xylene, they were rehydrated in 100%, 95%, and 75% ethanol. Free thiol groups were then blocked using a buffer that contained 25 mM 4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid, pH 7.4, 0.1 mM EDTA, pH 8.0, 0.01 mM neocuproine (Sigma-Aldrich, N1501), 40 mM N-ethylmaleimide (Sigma-Aldrich, E1271), and 1% Triton (Sigma-Aldrich) for 30 min. After three washes in PBS, S-glutathionylated cysteine groups were reduced by incubation with 13.5 μg/mL human Grx1 (Fitzgerald, 30R-1244), 35 μg/mL GSSG reductase (Roche, 10105678001), 1 mM GSH (Sigma-Aldrich, G4251), 1 mM NADPH (Roche, 10107824001), 18 μmol EDTA, and 137 mM Tris·HCl, pH 8.0, for 20 min. After three washes with PBS, newly reduced cysteine residues were labeled with 1 mM 3-(N-maleimidylpropionyl)biocytin (MPB) (Santa Cruz, sc-216373) for 1 h. Excess MPB was removed by three washes with PBS. Next, tissue samples were incubated with 0.5 μg/mL streptavidin-conjugated Alexa Fluor 488 (Invitrogen, S32354) for 30 min. Nuclei were stained with DAPI. All steps were conducted at room temperature. Slides were photographed under an Olympus BX61 confocal microscope.

Grx1 activity was assayed as described previously^{73 –75 (link)}. Briefly, lung tissue was lysed in 137 mM Tris-HCl, pH 8.0, 130 mM NaCl, and 1% NP-40. Lysates were then cleared by centrifugation and 100 μg was incubated with reaction mixtures containing Na/K phosphate buffer (0.1 mM, pH 7.5), 0.5 mM GSH (Sigma-Aldrich, G4251), 2 units/mL GSSG reductase (Sigma-Aldrich, G3664), 0.1 mM L-CySSG (Cayman, 17582), 0.2 mM NADPH (Roche, 10107824001) and 1.5 mM EDTA (pH 8.0) or with reaction buffer consisting of 137 mM Tris-HCl buffer (pH 8.0), 0.5 mM GSH (Sigma-Aldrich, G4251), 1.2 units GSSG reductase (Roche, 10105678001), 2.5 mM 2-hydroxyethyl disulfide (Sigma-Aldrich, 380474), 0.35 mM NADPH (Roche, 10107824001), 1.5 mM EDTA (pH 8.0). The reaction was prepared in a 96-well plate (final volume of 200 µL) and proceeded at 30 °C. The consumption of NAPDH was followed spectrophotometrically at 340 nm. In each sample, the spontaneous consumption of NADPH was subtracted. Data are expressed in units (U)/mg protein for lung homogenates in which 1 U equals the oxidation of 1 μmol NADPH/min.