Nadph

Glutathione peroxidase (GPx, EC 1.11.1.9) activity was evaluated by the decrease of NADPH (Sigma Chemical Co., St. Louis, MO, USA) concentration at 340 nm. The reaction medium contained 100 mM potassium phosphate buffer (Sigma Chemical Co., St. Louis, MO, USA), pH 7.7, 1 mM EDTA (Sigma Chemical Co., St. Louis, MO, USA), 2 mM reduced glutathione (Sigma Chemical Co., St. Louis, MO, USA), 0.15 U/mL glutathione reductase (Sigma Chemical Co., St. Louis, MO, USA), 0.4 mM azide (Sigma Chemical Co., St. Louis, MO, USA), 0.1 mM NADPH, and 0.5 mM tert-butyl hydroperoxide (Sigma Chemical Co., St. Louis, MO, USA) as enzyme substrate. GPx unit is defined as 1 µmol of NADPH consumed per minute and the specific activity as units/mg protein [42 (link)].

HDFs were lysed with the lysis buffer (20 mM Hepes, pH 7.2, 1% Triton X-100, 150 mM NaCl, 0.1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM EDTA, and 1 μg/mL aprotinin). After incubation for 30 min at 4°C, cellular debris was removed by centrifugation at 10,000 ×g for 30 min. NADPH oxidase in the supernatant was measured by lucigenin chemiluminescence in the presence of 500 μM NADPH (Sigma-Aldrich, St. Louis, MO) and 25 μM lucigenin (Sigma-Aldrich, St. Louis, MO) as described previously [16 (link)].

N27 cells were collected in krebs-hepes buffer and homogenized. The resultant suspension was assayed for protein concentration using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). NADPH-oxidase activity was measured in 30 μg of extracted proteins in the presence of lucigenin (5 × 10⁻⁶ mol/L; Sigma) and NADPH (10^− 4 mol/L; Sigma). Measurements were performed with an Infinite M200 multiwell plate reader (Tecan, Switzerland). No enzymatic activity was detected in the absence of NADPH.

Activity assays for wild-type α₂ and the Gln294Ala and Arg298Ala mutants were performed using a continuous, coupled, spectrophotometric assay monitoring the consumption of NADPH by the Trx/TrxR system (Ge et al., 2003 (link)). All experiments were performed on a Cary Bio300 spectrometer (Agilent) with data analysis performed using the Cary WinUV Kinetics program (Agilent) and Microsoft Excel. The assay buffer consisted of 50 mM HEPES pH 7.6, 15 mM MgCl₂, 1 mM EDTA, and the following substrate and effector concentrations were used: 1 mM CDP and 3 mM ATP (control for presence of activity), 1 mM CDP and 175 μM dATP (control for inactivation by dATP), 1 mM ADP and 120 μM dGTP, 1 mM GDP and 250 μM TTP, and 1 mM CDP/UDP and 1 μM dATP. Substrate, effector, E. coli Trx (30 μM), E. coli TrxR (0.5 μM), and NADPH from Sigma-Aldrich (200 μM) were mixed in assay buffer, and the reaction was initiated by the addition of α₂ (0.1 μM) and wild-type β₂ (1 μM) to a final volume of 120 μL. The decrease in NADPH absorbance at 340 nm was monitored over 1 min. The basal level of NADPH oxidation was monitored over 30 s prior to the addition of enzyme.