Cell-free NADPH oxidase activation assays: "in vitro veritas"

The superoxide (O₂^•- )-generating NADPH oxidase complex of phagocytes comprises a membraneimbedded heterodimeric fl avocytochrome, known as cytochrome b₅₅₈ (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 specifi c 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 "fl ow" along a redox gradient, from NADPH to oxygen, leading to the one-electron reduction of molecular oxygen to O ₂^•- . Methodological diffi culties 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 and the cytosolic components and the resulting O₂ ^•- generation. Cell-free assays consist of a mixture of the individual components of the NADPH oxidase complex, derived from resting phagocytes or in the form of purifi ed recombinant proteins, exposed in vitro to an activating agent (distinct from and unrelated to whole cell stimulants), in the presence of NADPH and oxygen. Activation is commonly quantifi ed by measuring the primary product of the reaction, O₂^•- , trapped immediately after its generation by an appropriate acceptor in a kinetic assay, permitting the calculation of the linear rate of O ₂^•- production, but numerous variations exist, based on the assessment of reaction products or the consumption of substrates. Cell-free assays played a paramount role in the identifi cation and characterization of the components of the NADPH oxidase complex, the deciphering of the mechanisms of assembly, the search for inhibitory drugs, and the diagnosis of various forms of chronic granulomatous disease (CGD).