Endothelial nitric oxide synthase (eNOS) plays an important role in the regulation of vascular tone and platelet aggregation. The principal investigator has been able to obtain mg amounts of wild-type and mutant eNOS from bacculovirus-infected Sf9 cells and has characterized the enzymatic properties and posttranscriptional modification of the WT protein as well as the interactions of the WT and mutant protein with lipids. The goal of the proposal is to extend the preliminary studies to gain a detailed understanding of how eNOS is regulated by these posttranslational modifications and interactions with acidic phospholipids and calmodulin. The principal investigator will use a combination of biochemical and molecular biological approaches to address these questions. Site directed mutagenesis will be used to examine the role of basic residues in the eNOS CaM-binding domain and potential sites of palmitoylation in promoting association with the cell membrane. Regulation of enzyme activity and/or association with acidic membrane lipids by phosphorylation will be investigated by 32P-labeling of endothelial cells in vivo and the kinase specificity investigated using available purified protein kinases in vitro. Chimeric constructs of eNOS and the mainly cytosolic inducible NOS (iNOS form) will be used to explore the molecular basis of potential differing interactions of these proteins with calmodulin and acidic phospholipids. Finally, experiments using sucrose gradient centrifugation and gel filtration chromatography will be used to explore possible differences in subunit number between eNOS, thought to function as a monomer, and iNOS which requires dimerization for expression of catalytic activity. Basic knowledge from these experiments should provide important insights into eNOS regulation and structure which may be useful in elucidating the molecular defects that lead to altered vascular endothelial NO production in human disease states such as atherosclerosis, diabetes and hypertension.
