Stimulation of human neutrophils induces a """"""""respiratory burst"""""""" causing production of 02- and H202. This response is crucial to normal bacterial killing but an inappropriate response contributes to neutrophil-mediated tissue damage in many diseases. Stimulation of the respiratory burst involves activation of an electron transport complex, the NADPH oxidase. Mechanisms of oxidase activation are largely undefined. Even the components of the oxidase are controversial, although data strongly support that it at least involves an NADPH-binding flavoprotein and a cytochrome. The cytochrome is being intensively studied elsewhere, but the flavoprotein has not been purified in quantity and its mechanisms of activation and electron transfer are unknown. This project will complete the development of a novel NADPH- affinity method for purification of the NADPH-binding oxidase flavoprotein. It will also employ new methods for assessing purification: 1) The ability to form a borohydride-reducible adduct between the protein and 2,3-dialdehyde-NADPH. 2) The ability to reconstitute NADPH-dependent 02- production in liposomes. For this model, flavoprotein purification fractions will be combined with solubilized membrane (the membrane being specifically depleted of functional NADPH-binding flavoprotein but providing all other components of the oxidase complex), for co- reconstitution of all oxidase components in liposomes by removal of detergent. Redox couples (FAD, FMN, thiols, iron-sulfur, transition metal) within the flavoprotein we be determined. Spectral, fluorescent and electron paramagnetic resonance methods will be used for titration of the flavoprotein with NADPH versus dithionite to determine stoichiometry of NADPH to flavin electron transfer and formation of intermediate redox states. Parallel studies of flavoprotein from resting and stimulated cells will test the hypothesis that oxidase activation involves increased substrate affinity (anaerobic NADPH binding) or catalysis (electron transfer). Tryptic peptides derived from the protein will be sequenced. The sequences will allow synthesis of peptides for raising anti- flavoprotein antibodies and for synthesizing cDNA probes, each of which can be used for molecular cloning of the flavoprotein cDNA.
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