Attenuation of p47phoxand p67phoxMembrane Translocation as the Inhibitory Mechanism ofS-Nitrosothiol on the Respiratory Burst Oxidase in Human Neutrophils

Abstract

The effect of the S-nitrosothiol (RSNO) on the activation of NADPH oxidase in human neutrophils was studied using anin vitrotranslocation system in which an anionic amphiphil, such as sodium dodecyl sulfate or arachidonate, plays a role as an activator. When membranes pretreated with RSNO and a cytosol fraction from resting neutrophils were combined to reconstitute the NADPH oxidase, both translocation of the cytosolic NADPH oxidase components such as p47phoxand p67phoxto the plasma membrane fraction and subsequent superoxide generation was inhibited. However, RSNO had no effect on O2−production when added after enzyme activation. A similar inhibition of translocation of recombinant p47phoxwas observed with RSNO-treated membrane. When the RSNO-treated membrane fraction was exposed to 2-mercaptoethanol the inhibition was reversed. The data suggest that RSNO inhibits translocation of p47phoxor p47phoxcontaining cytosolic complex via a direct effect on the membrane component of the NADPH oxidase.