Oxygen-derived radicals such as superoxide and its secondary products participate in a variety of normal and pathological processes. Neutrophil-derived oxygen reduction products participate normally in host defense against microbial infection, but have also been implicated in damage to host tissue in reperfusion injury (e.g. after restoration of blood flow to a myocardial infarct), shock lung, chronic inflammatory diseases (rheumatoid arthritis, Crohn's disease, etc.). the enzymatic system which catalyzes oxygen radical generation is the superoxide- generating respiratory burst oxidase or NADPH-oxidase. The enzymology and regulation of this enzyme system is poorly understood, in part due to the extraordinary instability of the system, and it is now thought to be comprised of several subunits including a flavoprotein and a heme protein. The proposed studies will investigate the enzymology and regulation of the oxidase using a variety of approaches. We will initially explore stabilization strategies, including a promising new approach, chemical cross-linking with reversible and irreversible cross- linkers, and will use the reversible cross-linker to investigate the subunit structure of the activated oxidase in the plasma membrane using a """"""""nearest neighbor analysis:. Antibodies to various known or suspected components of the oxidase will be prepared, and characterized, and used for a variety of approacher, including affinity isolation of the oxidase. Antibodies to one of these components will aid in the molecular cloning and sequencing of the cDNA of the FAD-containing component. Antibodies and Immunochemical techniques will be used along with new separation methods to investigate the location and translocation off of oxidase components in resting va. activated neutrophils. Isolated enzyme will be characterized by kinetic and immunochemical methods.
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