In response to a variety of stimuli, human neutrophils undergo a respiratory burst in which oxygen is converted to superoxide (O2-), hydrogen peroxide, and hydroxyl radicals (or related radical species). While these agents generally serve a beneficial role in killing ingested microorganisms, they also can cause serious damage to normal tissues during inflammation. In addition, several human diseases are associated with failure to appropriately regulate oxygen radical production. In adult respiratory distress syndrome, pulmonary damage occurs due to excessive oxygen radical production while in chronic granulomatous disease (CGD), failure to produce these radicals leads to life-threatening infections. The studies proposed are part of a long range goal to elucidate the biochemical mechanism which regulates the enzyme responsible for generating oxygen radicals in phagocytes, NADPH oxidase. Two new methods have been developed in this laboratory for studying this problem. The first allows the activation of NADPH oxidase to be studied in disrupted cells. Dormant oxidase present in the membranes from unstimulated human neutrophils can be activated with arachidonic acid provided that a previously unrecognized cytosolic factor is also present. In the second method, membranes from unstimulated cells can be solubilized in such a way that they retain their ability to be activated by arachidonate and cytosolic factor. Since all of the components necessary for NADPH oxidase activation in the cell-free system can now be rendered soluble by these two methodologies, the major goal of this research grant will be to purify an characterize the cytosolic and membrane components required for NADPH oxidase activation. To accomplish this overall objective, four specific aims have been identified: 1) to purify the cytosolic factor and then raise both polyclonal and monoclonal antibodies to the purified material; 2) to determine the identity of the cytoplasmic factor and the mechanism by which it participates in the activation of NADPH oxidase; 3) to determine the membrane components required for the activation of NADPH oxidase as well as its catalytic activity; and 4) to identify the molecular lesions in the three major genetic forms of chronic granulomatous disease. These studies might ultimately lead to new pharmacological methods for controlling oxygen radical-mediated tissue damage as well as to an improved understanding of the pathophysiology of immune disorders like CGD.
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