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Published online before print July 14, 2006

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(Journal of Leukocyte Biology. 2006;80:630-639.)
© 2006 by Society for Leukocyte Biology

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

Itai Pessach^*, Zeev Shmelzer^*, Thomas L. Leto, Mary C. Dinauer and Rachel Levy^*,1

^* Infectious Diseases Laboratory, Department of Clinical Biochemistry, Faculty of Health Sciences, Ben-Gurion University of the Negev and Soroka Medical Center, Beer Sheva, Israel;
Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland; and
Herman B. Wells Center for Pediatric Research and Department of Pediatrics (Hematology/Oncology), James Whitcomb Riley Hospital for Children, Indiana University School of Medicine, Indianapolis

¹ Correspondence: Department of Clinical Biochemistry, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer Sheva 84105, Israel. E-mail: ral{at}bgumail.bgu.ac.il

We have previously established a model of cytosolic phospholipase A₂ (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.

Key Words: FAD • arachidonic acid • cPLA₂