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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI022809-07
Application #
3134354
Study Section
Physical Biochemistry Study Section (PB)
Project Start
1989-09-30
Project End
1994-07-31
Budget Start
1992-08-01
Budget End
1993-07-31
Support Year
7
Fiscal Year
1992
Total Cost
Indirect Cost
Name
Emory University
Department
Type
Schools of Medicine
DUNS #
042250712
City
Atlanta
State
GA
Country
United States
Zip Code
30322
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Nisimoto, Y; Freeman, J L; Motalebi, S A et al. (1997) Rac binding to p67(phox). Structural basis for interactions of the Rac1 effector region and insert region with components of the respiratory burst oxidase. J Biol Chem 272:18834-41
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Ogata, K; Nishimoto, N; Uhlinger, D J et al. (1996) Spermine suppresses the activation of human neutrophil NADPH oxidase in cell-free and semi-recombinant systems. Biochem J 313 ( Pt 2):549-54
Kreck, M L; Freeman, J L; Abo, A et al. (1996) Membrane association of Rac is required for high activity of the respiratory burst oxidase. Biochemistry 35:15683-92
Nisimoto, Y; Otsuka-Murakami, H; Lambeth, D J (1995) Reconstitution of flavin-depleted neutrophil flavocytochrome b558 with 8-mercapto-FAD and characterization of the flavin-reconstituted enzyme. J Biol Chem 270:16428-34
Uhlinger, D J; Tyagi, S R; Lambeth, J D (1995) On the mechanism of inhibition of the neutrophil respiratory burst oxidase by a peptide from the C-terminus of the large subunit of cytochrome b558. Biochemistry 34:524-7

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