Arachidonic acid increases the activity of the assembled NADPH oxidase in cytoplasmic membranes and endosomes

Abstract

The effect of arachidonic acid (AA) on the assembled NADPH oxidase activity in cytoplasmic membranes and in endosomes separated from human neutrophils was studied. These two fractions were separated on a Percoll-sucrose density gradient from PMA-stimulated neutrophils preincubated with fluorescein isothiocyanate-conjugated dextran (FITC-dextran). In both fractions, NADPH oxidase activity could be detected with the addition of NADPH and cytochrome c, indicating the presence of an assembled activated form of the enzyme. Addition of AA at low concentrations (ED50 = 1 microM and 0.1 microM for cytoplasmic membranes and FITC-dextran endosomes, respectively) caused an increase in the activity of the assembled NADPH oxidase found in these fractions. Addition of 10 microM AA to the assembled oxidase in cytoplasmic membranes or endosomes significantly increased the Vmax (1.37 and 1.45 nmol O2/min compared with 2.05 and 2.20 nmol O2/min in the absence of presence of AA, respectively) and lowered the Km for NADPH (35 microM and 40 microM compared with 7.5 microM and 7.2 microM in the absence or presence of AA, respectively). These results suggest that AA increases the activity of the assembled NADPH oxidase by elevating the number of its active forms and increasing its affinity to the substrate.