Journal: Redox Biology

Article Title: Cytochrome b 5 reductase is the component from neuronal synaptic plasma membrane vesicles that generates superoxide anion upon stimulation by cytochrome c

doi: 10.1016/j.redox.2017.11.021

Figure Lengend Snippet: Correlation between NADH oxidation, O 2 consumption and superoxide anion production by soluble C b 5 R. Panel A : Representative traces of the NADH oxidation by soluble C b 5 R (2.5 and 5 μg/mL) are shown. NADH oxidase activity was measured from absorbance decay at 340 nm at 37 °C, in the following assay medium (pH 7.0): potassium phosphate 20 mM, DTPA 0.1 mM, NADH 100 μM in presence of soluble C b 5 R 2.5 (black line) and 5 μg/mL (grey line). Dotted lines indicate the slopes used to calculate the activity. Panel B : Oxygen consumption kinetics for soluble C b 5 R 1 μg/mL (black line) 5 μg/mL (grey line). The reaction was started by addition of C b 5 R at the time marked by a large drop of trace signals. O 2 consumption was measured in presence of soluble C b 5 R using a Oxygraph Plus DW1 (Hansatech Instruments) electrode, filled with 2 mL of the assay medium indicated in the Panel A. Dotted lines indicate the slopes used to calculate the activity. Panel C: O 2 ·- production by soluble C b 5 R was measured with NBT. Representative traces for the kinetics of NBT reduction by soluble C b 5 R isoform, and sensitivity to SOD is shown. NBT reduction was measured at 37 °C at 560 nm with soluble C b 5 R 2.5 μg/mL in absence (black line) or presence of SOD 1 U/mL (dotted line) in the assay medium indicated in the Panel A, supplemented with NBT 200 μM. The dotted line indicates the slope used to calculate the activity. Panels D, E and F : Dependence upon C b 5 R concentration for soluble (filled squares) and membrane (open squares) C b 5 R, respectively, of NADH oxidation, oxygen consumption and superoxide production. All the results shown in this Figure are the average (± standard errors) of triplicate experiments.

Article Snippet: Panel B : O 2 consumption kinetics at 37 °C for membrane C b 5 R 1 μg/mL (black line) 5 μg/mL (grey line) measured with an Oxygraph Plus DW1 (Hansatech Instruments) electrode, filled with 2 mL of the assay medium indicated in Panel A.

Techniques: Activity Assay, Concentration Assay