Nitric oxide (NO) biosynthesis has emerged as an important factor in human health and diseae. NO generated in small amounts appears to serve a signaling facedown, whereas production of excessive amounts is linked to numerous diseases including sepsis, immune-type diabetes, inflammatory bowel disease, rheumatoid arthritis, carcinogenesis, multiple sclerosis, and transplant rejection. Immunostimulation induces expression of a distinct NO synthase isoform (iNOS) in many tissues, including lung, liver, kidney, heart, smooth muscle, and intestine. Because iNOS generates large amounts of NO, its expression has become increasingly linked to the diseases noted above. We are studying the biochemistry of mouse macrophage iNOS, which is highly homologous to human iNOS. Macrophage iNOS is a bi-domain enzyme that contains FAD, FMN, tetrahydrobiopterin, heme, and calmodulin. In macrophages and other cells, iNOS is expressed as a mixture of monomers and dimers, with the monomer being inactive regarding NO synthesis. We hypothesize that dimerization of iNOS is a key determinant in its activation, and may serve as a physiologic and/or pharmacologic control point for iNOS function within cells. We will test this hypothesis with four specific experimental objectives; First, we will determine if dimerization physically alters the heme environment, thus affecting its reactivity, or if dimerization unmasks binding sites for H4biopterin and L- arginine in the oxygenase domain, thus creating the enzyme active site. Techniques include a variety of spectroscopies, redox potentiometry, and radioligand binding. Secondly, we will investigate whether dimerization enables the iNOS reductase and oxygenase domains to communicate electronically in a productive manner. Techniques include visible spectroscopy and creation of iNOS heterodimeric structures composed of non- identical subunits that differ with regard to domain composition or amino acid sequence. Thirdly, we will seek to identify the specific protein regions involved in subunit dimeric interaction and cofactor binding within the iNOS oxygenase domain. This will involve testing oxygenase domain peptides and fragments for H4biopterin, heme, and L-arginine binding, determining their ability to form dimers, or antagonize dimerization; mapping the oxygenase domain with monoclonal antibodies raised against iNOS; examining oxygenase domain mutants for dimerization, cofactor binding, and catalytic function; and crystallization of the dimeric oxygenase domain. Lastly, we will investigate whether iNOS subunit dimerization is an important physiologic or pharmacologic control point in cells, by monitoring dimer formation in cells over time, relating it to levels of cellular factors thought to promote dimerization, and testing whether dimerization can be manipulated pharmacologically. Together, this will provide a comprehensive picture regarding how dimer formation activates iNOS, and how it can be controlled.
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