Regulation of superoxide-producing NADPH oxidases in nonphagocytic cells

The membrane-integrated protein gp91^phox functions as the catalytic center of the superoxide-producing phagocyte NADPH oxidase. Recent studies have identified homologs of gp91^phox in nonphagocytic cells, which constitute the NADPH oxidase (Nox) family. Activation of the Nox oxidases leads to production of reactive oxygen species (ROS), thereby participating in a variety of biological events, such as host defense, hormone biosynthesis, and signal transduction. The activity of the Nox enzymes is regulated by various proteins, including the small GTPase Rac; regulatory mechanisms differ dependent on the type of the Nox proteins. For example, an oxidase activator (p47 ^phox or Noxo1) and an oxidase activator (p67^phox or Noxa1) are absolutely required for superoxide production by gp91^phox and Nox1, but not by Nox3. Rac, albeit probably dispensable to the Nox3 activity, plays an essential role in activation of gp91^phox. Thus, functional reconstitution of Nox systems is crucial for the study of Nox regulation. Here we describe a basic method for the reconstitution of Nox systems by expression of oxidase proteins in transfectable cells.