The long term objective of this proposal is to determine the molecular basis for superoxide production in human neutrophils. The proposed investigation will focus on the role of human neutrophil cytochrome b-559 in the delivery of reducing equivalents to molecular oxygen at the sites of inflammation. Understanding the molecular mechanism of this delivery and its regulation will provide crucial information necessary for understanding, at the molecular level, the microbicidal killing and misdirected tissue injury functions of human neutrophils. In addition, it will aid in laying the foundation for an ultimate genetic cure of chronic granulomatous disease. More specifically, this proposal outlines strategies for: 1) determination of unknown structural parameters of cytochrome b559 such as amino acid sequence of one of the subunits (22 kilodalton light chain), localization of the heme prosthetic group, relationship of the molecule to the membrane, and structural changes accompanying activation of superoxide production; 2) biochemical, immunological, and biophysical determination of the requirement for cytochrome b-559 as the terminal component of the superoxide generating system in the neutrophil; 3) determination of the distribution, mobility, and biochemical state of the cytochrome b-559 in resting and activated human neutrophils to test hypotheses concerning its mechanism of action and functional role in tissue injury; 4) reconstitution of superoxide production using isolated and purified b-cytochrome and other components of the neutrophil in order to create a model system to study plasma membrane electron transport required for superoxide production; 6) production additional immunological and biochemical probes of structure and function to facilitate the molecular dissection of neutrophil superoxide production. Potentially this work will contribute to understanding how the superoxide generating system can be externally manipulated to enhance host defenses but reduce inflammation at potentially accessible sites such as lung tissue.
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