NADpH oxidoreductase

NADPH oxidase (NOX) is a membrane bound enzyme present in neutrophils and other phagocytic cells (138). This plasma membrane associated enzyme complex catalyzes the univalent reduction of oxygen using NADPH as an electron donor to produce superoxides, which are involved in the bactericidal activity of the neutrophils (137,138). Though NOX is involved in the generation of ROS for bactericidal activities, recent research has shown that a new family of NADPH oxidases are expressed in various cell types, including the epithelium, smooth muscle cells, and the endothelium (134,135). These enzymes produce superoxide and other ROS, including hydrogen peroxide (Figure 10.1). The activity of this enzyme in vascular cells is a main source of ROS in inflammation and is believed to be a cause for atherosclerosis and hypertension. NOX expression is also found to be high in certain forms of cancer and endocrine disorders as a result of superoxide and other ROS production (137-139). Elevated levels of ROS are also seen in patients with progressive neurological diseases like Alzheimer's, and Parkinson's disease (140). These recent discoveries about the many different functions of the nonphagocytic functions of NOX has made it an attractive target for therapeutic agents and an important biomarker for these diseases in different cell types (139,140). NOX is usually measured in cell free extracts by using them with NADPH for superoxide radical generation, which is measured by following the reduction of ferric cytochrome-C at 550 nm (141,142) or by monitoring the inhibition of the formation of formazan blue from NBT at 560 nm (143). Use of a new tetrazolium based assay to study the production of superoxide radicals has also been done, with the NOX and

NADPH as a source for superoxide radical generation (143). Chemiluminescence assays based on oxidation of a chromophore such as lucigenin are also being used to measure NOX activity (144). In this assay, the chromophore becomes chemiluminescent after reacting with the superoxide radical generated from the NOX reaction. This is then measured and quantitated as NOX activity (145).

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