The C-terminal flavin domain of gp91^phox bound to plasma membranes of granulocyte-like X-CGD PLB-985 cells is sufficient to anchor cytosolic oxidase components and support NADPH oxidase-associated diaphorase activity independent of cytosolic phospholipase A₂ regulation

We have previously established a model of cytosolic phospholipase A ₂ (cPLA₂)-deficient PLB-985 cells and demonstrated that cPLA₂-generated arachidonic acid (AA) is essential for reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation and NADPH-dependent diaphorase activity. The present study focuses on the C-terminal cytoplasmic domain of gp91^phox (residues 283-570), which contains the NADPH binding and flavin adenine dinucleotide-reducing center, to determine if this portion is regulated by AA. The gp91^phox C-terminal reductase domain was expressed in X-CGD PLB-985 cells lacking normal gp91^phox (X-CGD PLB 91CT cells) and was detected in the plasma membrane. It appears to be bound electrostatically to the plasma membrane, as it is eluted by high salt. Permeabilized, granulocyte-like X-CGD PLB 91CT cells lacking cPLA₂ protein and activity, as well as AA release after stimulation, supported NADPH-dependent diaphorase activity after stimulation, similar to granulocyte-like X-CGD PLB 91CT cells. Normal translocation of p47 ^phox and p67^phox to the membrane fractions of both stimulated cell types indicated that the gp91^phox C-terminal region is sufficient to anchor the cytosolic oxidase components to the membranes. cPLA₂ translocated to membranes and bound the assembled oxidase in granulocyte-like X-CGD PLB 91CT cells after stimulation. Therefore, the assembled membrane-bound oxidase complex encompassing the flavin domain of gp91^phox provides a docking site for cPLA₂ but is not the site of AA-based regulation of oxidase activity.