REGULATION OF OXYGEN METABOLISM IN BLOOD NEUTROPHILS

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The production of oxygen radicals by human blood neutrophils is critical for the success of host defense, but also may be detrimental to the host in certain inflammatory processes, i.e., in the adult respiratory distress syndrome, arthritis, etc. Understanding the regulation of oxygen radical production is the long-term goal of this project and is important for developing methods of controlling various disease states. The enzyme in neutrophils which generates oxygen radicals is a membrane-bound, multi- component electron transport chain, termed NADPH oxidase, and recently shown to consist of at least four-five polypeptides. The mechanisms of activation of this complex and the exact functions of its components are still unclear, but activation may involve both phosphorylation reactions and assembly of components. One phosphoprotein component of NADPH oxidase, a 47 kDa protein (pp47), has been identified, but the functional role of its phosphorylation is unknown. Specific Aim 1 of this application propose to examine the role of phosphorylation reactions in the functions and regulation of pp47 and other oxidase components. Using a novel assay for oxidase assembly, based on a newly-developed immunobead technique, the participation of phosphorylation reactions by both serine/threonine and tyrosine protein kinases in the assembly and activation of NADPH oxidase will be analyzed. In addition, cell-free systems will be utilized to study the interactions between specific protein kinase and recombinant and purified native oxidase components. A large body of data implicates protein kinase C (PKC) as an important regulator of NADPH oxidase, but the mechanisms involved are unknown. It has recently been recognized that PKC consists of a family of isozymes, which are differentially distributed and regulated in different cell types, and we have identified at least two PKC isozymes in human neutrophils. Specific Aim 2 of this application proposes a comprehensive characterization of the PKC family in these cells in order to understand the biological roles of individual isozymes in the activation of NADPH oxidase. Using specific antibodies and biochemical analysis, the PKC isozymes in human neutrophils will be identified, separated, and their regulatory and autophosphorylation properties characterized. The separated isozymes will be utilized in cell-free phosphorylation systems with NADPH oxidase components. Overall, these studies should provide new insights into the biochemical regulation of the respiratory burst.