Cell-Free NADPH Oxidase Activation Assays: “In Vitro Veritas”

The superoxide (O2 ∙−)-generating NADPH oxidase complex of phagocytes comprises a membrane-imbedded heterodimeric flavocytochrome, known as cytochrome b 558 (consisting of Nox2 and p22 phox ) and four cytosolic regulatory proteins, p47 phox , p67 phox , p40 phox , and the small GTPase Rac. Under physiological conditions, in the resting phagocyte, O2 ∙− generation is initiated by engagement of membrane receptors by a variety of stimuli, followed by specific signal transduction sequences leading to the translocation of the cytosolic components to the membrane and their association with the cytochrome. A consequent conformational change in Nox2 initiates the electron “flow” along a redox gradient, from NADPH to oxygen, leading to the one-electron reduction of molecular oxygen to O2 ∙−. Methodological difficulties in the dissection of this complex mechanism led to the design “cell-free” systems (also known as “broken cells” or in vitro systems). In these, membrane receptor stimulation and all or part of the signal transduction sequence are missing, the accent being placed on the actual process of “NADPH oxidase assembly,” thus on the formation of the complex between cytochrome b 558 an...
Source: Springer protocols feed by Infectious Diseases - Category: Infectious Diseases Source Type: news