Fig. 6. Inhibitory effect of DC membranes on the elicited neutrophil NADPH oxidase activity in a cell-free system. NADPH oxidase activity (nmol O₂^– produced per minute in each well of the microtiter plate) was determined after preincubation for 10 minutes at room temperature of bovine neutrophil membranes (6 µg protein/assay) or murine DC membranes (10.5 µg protein/assay) or a mixture of neutrophil membranes (6 µg protein/assay) and DC membranes (10.5 µg protein/assay) with neutrophil cytosol (60 µg protein), ATP, GTPS, MgSO₄, and increasing amounts of arachidonic acid (AA) (final volume 30 µl). The preincubation step was followed by addition of cytochrome c, KCN and NADPH in PBS (see Materials and Methods). The figure shows the rate of production of O₂^– per minute at different concentrations of arachidonic acid. (A) Neutrophil membranes (); DC membranes (); neutrophil membranes mixed with DC membranes () (the shift of the optimal concentration of arachidonic acid towards higher values when DC membranes and neutrophil membranes are added together in the medium is due to the higher levels of membrane lipids functioning as an unspecific trap of arachidonic acid). (B) Dose-response of the inhibitory effect of DC membranes on the neutrophil oxidase activity at the optimal concentration of arachidonic acid (); DC membranes heated for 10 minutes at 100°C and mixed with neutrophil membranes (); DC membranes treated for 1 hour at 20°C with proteinase K (proteinase K/membrane protein: 1/100) and mixed with neutrophil membranes ().