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   This is assayed in a cell-free system by measuring the superoxide-dependent reduction of cytochrome c, monitored spectrophotometrically. Cell-free activation is initiated with an anionic amphiphile such as arachidonate, SDS or phosphatidic acid. Diacylglycerol synergizes in the activation, and GTP[S] is included to convert Rac into an active conformation. Although arachidonate and phosphatidic acid have both been proposed to be physiologically relevant activators of superoxide generation, their intracellular roles in NADPH oxidase regulation remain areas of investigation. Cytosol plus plasma membrane can be used as a source for the required factors, or the purified factors (mostly recombinant) can be used in a membrane-reconstituted system.

Lambeth, J.D. (1989) "Activation of the Respiratory Burst Oxidase in Neutrophils: On the Role of Membrane-Derived Second Messengers, Ca++ and Protein Kinase C" J. Bioenergetics and Biomembranes 20, 709-733.

Burnham, D.N., Uhlinger, D.J., and Lambeth, J.D. (1990) "Diradylglycerol Synergizes with an Anionic Amphiphile to Activate Superoxide Generation and Phosphorylation of p47phox in a Cell-free System from Human Neutrophils" J. Biol. Chem. 265, 17550-17559.

Tyagi, S.R., Neckelmann, N., Uhlinger, D.J., Burnham, D.N. and Lambeth, J.D. (1992) "Cell-free translocation of recombinant p47-phox, a component of the neutrophil NADPH oxidase: Effects of guanine 5'O-(3-thiotriphosphate), diacylglycerol, and an anionic amphiphile" . Biochemistry 31, 2765-2774.

Uhlinger, D.J., Burnham, D.N. and Lambeth, J.D. (1991) "Nucleoside Triphosphate Requirements for Superoxide Generation and Phosphorylation in a Cell-Free System from Human Neutrophils: SDS and Diacylglycerol Activate Independently of Protein Kinase C" J. Biol. Chem. 266, 20990-20997.