Mammalian synthesis of nitric oxide (NO) from L-arginine (L-Arg) is newly discovered and mechanistically novel. NO is synthesized in a wide variety of cells and tissues, where it acts as a signal or effector molecule in several physiological and pathological processes. These include control of blood pressure and blood flow, neuron signaling, chemotaxis, cell proliferation, thrombosis, and hepatic response to sepsis. Understanding the biochemistry of NO synthesis will greatly aid investigations into the function, distribution, and control of this metabolic pathway. Preliminary biochemical characterization of a partially-purified macrophage NO- generating activity shows it requires NADPH, tetrahydrobiopterin, FAD, and thiol in addition to L-Arg; and may be multi-component. The goal of this proposal is to purify, characterize, and clone the mouse macrophage enzyme(s). Purification will use conventional chromatographic resins (gel filtration, anion exchange) and affinity resins that are based on binding sites for L-Arg, NADPH, and FAD. The possible dissociation of an enzyme complex during chromatography will be addressed in each chromatographic step by mixing experiments. Diphenyleneiodonium (DPI), an irreversible inhibitor of NO production, will be synthesized using [125I]NaI and used to identify and purify the enzyme (or subunit) possessing NADPH/FAD binding sites. The enzyme(s) will be sequenced and cloned. Characterization studies will be done both during and following purification. They include determining the substrate specificity and Michaelis constants for L-Arg (and its analogs), NADPH, and DPI; the stoichiometric relationships between L-Arg-dependent NADPH oxidation, NO production and O2 consumption; and the source of the oxygens in the products (NO and the ureido oxygen of L-citrulline). To assist in determining the reaction mechanism, reaction conditions that lead to a buildup of intermediates will be sought and, if found, reaction intermediates will be isolated and their structure identified by NMR and gas chromatography-mass spectroscopy.
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