Fig. 2. Effect of H₂O₂ on cell-free NOX5 activity.

(A) Difference spectra of dithionite-reduced minus oxidized samples of crude membranes from K562 and K562/NOX5 cells (B) Superoxide production in crude membrane preparations. The assay was performed as described in Experimental Procedure using the Diogenes reagent in the presence of SOD (400 U/ml), DPI (10 μM), or sodium azide (1 mM), or in the absence of NADPH (−) or Ca²⁺ (−), as indicated. After normalization by subtraction of the zero-time value of the chemiluminescence output, the area under the curve was calculated as a measure of total superoxide production. *** P < 0.001 = statistical significance versus control K562 membranes. (C) Superoxide produced by K562/NOX5 membranes was determined in the presence of 100 μM H₂O₂ and increasing free Ca2+ concentrations. The values obtained are expressed as in B. No significant difference was observed versus the value obtained in the absence of H₂O₂. The data shown are the means +/− SEM of 3 to 7 independent experiments.