Ghaith Altawallbeh, Mohammad Mahfuzul Haque, Dennis J. Stuehr and Mekki Bayachou
Department of Chemistry, Cleveland State University, USA
Even though a lot is yet to be explored about eNOS structure-function relationship, its vital role in cardiovascular activities makes it one of the leading research targets today. eNOS is a complex redox protein, whose function harmonizes a group of partners including the cell membrane which modulates its activity. This enzyme and other related isoforms have been studied exclusively in solution despite evidence that membrane plays a role in at least the eNOS activity and how this affects its physiologic environment.
Therefore, we have incorporated the recombinant oxygenase subunit of the enzyme into miniature lipid membranes called nanodiscs. These lipid bilayers will provide a defined system to elucidate the influence of phospholipid bilayers on the structure and activity of eNOSoxy. The structural homogeneity and stability of nanodisc samples with and without eNOSoxy were determined using size exclusion chromatography, gel electrophoresis, and atomic force microscopy. The calculated stocks hydrodynamic diameter of both empty an eNOSoxy bound nanodiscs were 11.3 nm and 13.7 nm, respectively. The coomassie stained SDS-PAGE gel of selected fractions confirms the co-elution of the complex. The height measured by AFM for empty nanodiscs were ~5 nm consistent with the published data. The average size of nanodisc diameter was confirmed by 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 the enzyme drops by 2 folds when compared to the free form. These data suggest that the membrane environment affects the catalytic properties of eNOS heme domain.
Keywords: Nanodiscs, eNOSoxy, Cardiovascular, enzyme activity.