P101 – Endothelial Nitric Oxide Synthase (eNOS) on Lipid Nanodiscs: Toward A Soluble Assembly Reflecting Native-Like Function of eNOS

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Nitric Oxide


Cardiovascular disease (CVD) is the leading cause of death worldwide. Approximately 30% of all global deaths in 2008 were due to CVD. Endothelial cells cover the blood vessels lumen and provide a barrier against vascular disease. Nitric oxide is a unique bio-regulator with important signaling roles in cardiovascular as well as other physiologic systems. Nitric oxide synthases (NOSs) are a family of enzymes that generate nitric oxide from arginine and oxygen. Endothelial nitric oxide synthase (eNOS) is one member of this family, and is the dominant isoform in the inner walls of blood vessels. It regulates numerous essential cardiovascular functions including vasodilation (blood pressure), inhibition of platelet aggregation and adhesion to the vascular wall, which prevents atherosclerosis and unwanted blood clots. To determine the influence of the phospholipid bilayer on the structure and activity of eNOS in a defined system, we have incorporated the recombinant oxygenase subunit of the enzyme into miniature lipid membranes called nanodiscs which are 12.9 nm in diameter. These nanodiscs based on membrane scaffold proteins provide a unique system that mimics the enzyme's native microenvironment, yet the prepared enzyme/nanodisc assemblies can be conveniently studied in solution like any soluble enzyme preparation. Homogenous eNOS/nanodisc samples are purified using size exclusion chromatography. The average size of nanodisc diameter was confirmed by particle analysis based on dynamic light scattering. Griess assay is used to measure activity of free and nanodisc-bound enzymes. As compared to the free enzyme, the specific activity of nanodisc-bound eNOS oxygenase appears to be much lower. These data suggest that the membrane environment affects the catalytic properties of eNOS oxygenase.