Coupling of 6-phosphogluconate dehydrogenase with NADPH oxidase in neutrophils: Nox2 activity regulation by NADPH availability

It is well known that activation of the phagocyte NADPH oxidase requires the association of cytosolic proteins (p67-phox, p47-phox, p40-phox, and Rac) with the membrane cytochrome b558, leading to its conformation change. Recently, the phagocyte NADPH oxidase complex was isolated in a constitutively active form. In this complex, 6-phosphogluconate dehydrogenase (6PGDH), an enzyme involved in the production of intracellular NADPH, was identified. This protein was absent from the oxidase complex isolated from B lymphocytes, suggesting a specific interaction with the neutrophil NADPH oxidase. To clarify the implication of 6PGDH in the NADPH oxidase activity, a siRNA approach was conducted in neutrophil-like PLB985 cells. NADPH oxidase activity of siRNA-transfected cells was shown to be decreased. Similar results were obtained in vitro, after reconstitution of oxidase activity with subcellular fractions isolated from siRNA-transfected cells. Interestingly, the Michaelis constant (Km) of Nox2 for NADPH increas...