Neutrophils express on the surface of their plasmalemma when stimulated by contact with bacteria a NADPH:O2 oxidoreductase complex. Its enzymology is poorly understood. It exhibits intrinsic diaphorase activity in addition to superoxide and hydrogen peroxide Generating activity. Children born with cells unable to express oxidoreductase activity suffer from the fatal disease known as chronic granulomatous disease (CGD). In solubilized form the oxidoreductase dissociates into distinct catalytically inactive protomers: a cytosolic factor, a flavoprotein component, and a cytochrome b of unusual redox potential. Little is known regarding the properties of the flavoprotein and cytosolic factor. This proposal is aimed toward learning more about the properties and role of the flavoprotein in the oxidoreductase complex by purifying it to homogeneity, analyzing for its presence or absence in membrane preparations derived from patients with variant forms of CGD, and by reconstitution experiments designed to assess its role in conferring enzyme Activity to the oxidoreductase complex. Methods of purifying the flavoprotein to homogeneity are outlined. A method is proposed for labelling the NADPH binding site of the oxidoreductase to assess whether or not it resides with the flavoprotein or elsewhere in the assembled enzyme complex. Antibodies are to be prepared against the purified flavoprotein to test by immunoinhibition techniques the functional role of the flavoprotein in the assembled enzyme complex. The development of a simple method of analyzing for the flavoprotein by HPLC analysis is proposed as is exploratory work on the feasibility of developing an ELISA assay for the flavoprotein. The use of an NBT diaphorase type assay for classifying variant forms of CGD and identifying carriers of this disorder is also proposed. It is hypothesized that diaphorase activity arises in the oxidoreductase through association of the flavoprotein with the cytosolic factor whereas superoxide generating activity requires the additional presence of the cytochrome b component. Preliminary studies with the @diaphorase assay indicate that it may be useful in identifying CGD patients deficient in the cytosolic factor. An expanded study of the applicability of this latter assay in screening CGD patients, include% reconstitution mixing experiments, is proposed to evaluate the applicability and insight this test may offer in clarifying how the oxidoreductase is assembled into a catalytically active enzyme complex. The long range goal of this project is to define the enzymology of the oxidoreductase and to clarify how it is expressed in the neutrophil.
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