Gsh reductase

Cell lysates were collected and immediately subjected to protein thiol and GSH measurement as reported (41 (link), 42 (link), 43 (link)). Fluorescent intensities were detected at 400Ex/465Em by CLARIOstar microplate reader (BMG LABTECH). Quantitative determinations of GSH and GSSG levels were performed using the enzymatic recycling method. Briefly, protein in the cell extracts was precipitated by sulfosalicylic acid, and the supernatant was then divided into two parts. For reduced GSH, the supernatant was incubated with thiol fluorescent probe IV (Sigma; #595504), and fluorescence intensities were measured. For total GSH (GSH + GSSG), the supernatant was neutralized by triethanolamine and incubated with the reduction system, containing NADPH (Sigma; #10107824001) and GSH reductase (Sigma; #10105678001), at 37 °C for 20 min. GSSG was calculated based on the results from reduced GSH and total GSH; the ratio of GSH/GSSG = [GSH]/(([Total GSH] − [GSH])/2).

For GPx1 activity assay, PK15 cells were cultured at a density of 6 × 10⁴ cells/well in 12-well plates with corresponding treatment. The activity of GPx1 was measured with tert-butyl hydroperoxide (t-Bu-OOH) as substrate as described previously [29 (link)]. Briefly, cell extracts prepared by sonication (SonicsVCX105, USA) in ice-cold PBS was centrifuged at 12,000 rpm for 20 min to obtain the supernatant. Then, 50 μL of the supernatant was added to 1900 μL of the reaction mixture [50 mM Tris-HCl, pH 7.5, 2 mM EDTA, 2 mM GSH, 1 mM NaN3, 0.1 mM NADPH, and 0.9 U of GSH reductase (Sigma, USA)] and pre-incubated for 3 min at 25°C. Following this, 50 μL of t-Bu-OOH (8 mM) was added to the reaction mixture and the rate of NADPH oxidation was assessed spectrophotometrically by measuring the absorbance at 340 nm at 25°C for 5 min. The nonenzymatic reaction rate was determined by substituting water (serving as the blank) for the cell extracts. One unit of enzyme activity was defined as equivalent to 1 μmol of NADPH oxidized per minute under these conditions. GPx1 activity was expressed as % of the control values.

The biotin switch assay was performed following a method applied for cysteine modification detection of yeast ER Hsp70 Kar2 with some modifications (27 (link), 59 (link)). The supernatant of the cell lysis (3 ml) was mixed with 12 ml of urea-containing cysteine modification buffer (CMBU; 0.1 m HEPES-NaOH, pH 7.4, 1% SDS, 1 mm EDTA, 8 m urea) with cOmplete ULTRA protease inhibitor (Roche Applied Science) and 0.1 m NEM. Samples were placed for 30 min at room temperature (RT). Proteins were precipitated with 10% TCA on ice for 30 min. The pellet was collected by centrifugation and washed once with 5% TCA and twice with 70% acetone and dissolved in 300 μl of CMBU. Then 6 ml of Grx reduction buffer (0.1 m Tris-HCl, pH 8.0 containing 1 mm EDTA, 0.5 mm GSH, 1 mm NADPH (Sigma–Aldrich), 0.25 units/ml GSH reductase (Sigma–Aldrich) and 60 μg of purified E. coli Grx3 C14S/C65Y (27 (link), 33 (link), 38 (link), 44 (link), 64 (link)) or Grx3 C11S/C14S/C65Y (27 (link))) was added, and samples were incubated for 15 min at 37 °C. After reduction, samples were quenched with TCA, and protein precipitation was carried out as above. The pellet was dissolved in 300 μl of CMBU with 0.2 mm biotin-maleimide (Sigma–Aldrich) and was placed for 30 min at RT and then protein precipitation was carried out as above. The pellet was dissolved in 100 μl of CMBU.

GPx activity in lung homogenates (~4-8 μg) and cells (~50-70 μg) was measured via the hydrogen peroxide-dependent consumption of NADPH at 340 nm over 5 minutes in the presence of 0.2 mM NADPH, 1.5 mM reduced GSH, 1.0 mM sodium azide, 0.5 mM DTPA, GSH reductase (1 : 427, Sigma), and 1.6 mM hydrogen peroxide (Fisher) in 50 mM Tris-HCl buffer at pH 7.0.