The polymorphonuclear leukocyte (PMN) exerts its critical microbicidal effects in large part through a burst of oxidative metabolism mediated by an activatable NADPH oxidase that generates superoxide and other toxic oxygen derivatives. Recent studies in a reconstituted cell-free system have demonstrated the essential importance of two discrete cytoscolic proteins of 47 and 67 kDa in events leading to oxidase activation. Moreover, selective deficiency states of these proteins have been detected in tow different forms of autosomally inherited chronic granulomatous disease (CGD). These cytosolic oxidase components will be characterized in terms of structure and function. Structural studies will focus on the separation and purification of the 47 and 67 kDa proteins primarily by chromatographic and electrophoretic techniques. Monospecific polyclonal and monoclonal antibodies will be generated to the purified proteins and their potential immunocrossreactivity assessed. Structural analyses will proceed with proteolytic digestion, peptide mapping, amino acid analysis and sequencing. The structure and relatedness of other associated cytosolic proteins will e considered. Functional studies will begin with analysis of additional CGD patients to determine the prevalence of deficiency states for the 47 and 67 kDa proteins and to search for patients with deficiencies of other putative cytosolic oxidase components. Whether the 47 nd 67 kDa proteins exist as a non-covalently linked functional complex will be considered using native gels, velocity sedimentation and association with affinity matrices. Finally, experiments will be done to determine whether oxidase activation is associated with changes in the physical state of cytosolic proteins (e.g. translocation to a membrane-associated fraction) or with covalent modification (e.g.phosphorylation) of one or both of the proteins. These studies will provide a detailed structural and functional characterization of two newly described cytosolic components of the PMN NADPH oxidase system. The critical importance of these proteins is underscored by their absence from cells of patients with severe recurrent infections associated with autosomal varieties of CGD.

Project Start
Project End
Budget Start
Budget End
Support Year
1
Fiscal Year
1989
Total Cost
Indirect Cost
Name
University of Iowa
Department
Type
DUNS #
041294109
City
Iowa City
State
IA
Country
United States
Zip Code
52242
Britigan, B E; Rasmussen, G T; Olakanmi, O et al. (2000) Iron acquisition from Pseudomonas aeruginosa siderophores by human phagocytes: an additional mechanism of host defense through iron sequestration? Infect Immun 68:1271-5
McCormick, M L; Buettner, G R; Britigan, B E (1998) Endogenous superoxide dismutase levels regulate iron-dependent hydroxyl radical formation in Escherichia coli exposed to hydrogen peroxide. J Bacteriol 180:622-5
Miller, R A; Britigan, B E (1997) Role of oxidants in microbial pathophysiology. Clin Microbiol Rev 10:1-18
Olakanmi, O; Stokes, J B; Pathan, S et al. (1997) Polyvalent cationic metals induce the rate of transferrin-independent iron acquisition by HL-60 cells. J Biol Chem 272:2599-606
McCormick, M L; Metwali, A; Railsback, M A et al. (1996) Eosinophils from schistosome-induced hepatic granulomas produce superoxide and hydroxyl radical. J Immunol 157:5009-15
Nauseef, W M; Cogley, M; McCormick, S (1996) Effect of the R569W missense mutation on the biosynthesis of myeloperoxidase. J Biol Chem 271:9546-9
Miller, R A; Rasmussen, G T; Cox, C D et al. (1996) Protease cleavage of iron-transferrin augments pyocyanin-mediated endothelial cell injury via promotion of hydroxyl radical formation. Infect Immun 64:182-8
Andersen, K A; Britigan, B E; Wilson, M E (1996) Short report: regulation of inducible heat shock protein 70 genes in Leishmania chagasi. Am J Trop Med Hyg 54:471-4
Britigan, B E; Ratcliffe, H R; Buettner, G R et al. (1996) Binding of myeloperoxidase to bacteria: effect on hydroxyl radical formation and susceptibility to oxidant-mediated killing. Biochim Biophys Acta 1290:231-40
Edeker, B L; Rasmussen, G T; Britigan, B E (1995) Bicarbonate and phosphate ions protect transferrin from myeloperoxidase-mediated damage. J Leukoc Biol 58:59-64

Showing the most recent 10 out of 53 publications