The plasma lipoproteins (LPs) transport lipids between various extravascular compartments where they are utilized or stored. The high density LPs (HDL), very low density LPs (VLDL), and chylomicrons are modified in plasma by two enzymes. The free cholesterol of HDL is converted to its esterified form by lecithin:cholesterol acyltransferase (LCAT), an enzyme that also has phospholipase A2 activity; both processes are activated by apoA-I. Lipoprotein lipase (LPL) hydrolyzes the triglycerides of VLDL and chylomicrons, a reaction that is timulated by apoC-II. These enzymes are important in determining the distribution of steryl or fatty acyl groups among individual lipids and the distribution of these lipids among the plasma LPs. A molecular description of the activities and specificities of both enzymes is a key component of a thorough understanding of lipid metabolism since these activities determine the product structure, which affects their distribution inplasma. We propose a) to resolve the specificities of these enzymes into components that are dependent either upon the covalent structure of the individual substrate molecules or upon macroscopic properties such as fluidity or size, b) to measure the free energy of association (Delta Ga) of LCAT and LPL with phospholipids in the presence and absence of their apoprotein activators, and c) to determine the relationship between Delta Ga and the structure of the substrate and product LPs. These studies will be conducted with ether analogs of lipids, and a variety of hydrolyzable fluorescent or radioactive lipids. This investigation involves lipid synthesis, fluorescence and radioactive assays of LPL and LCAT activity, lipoprotein reassembly, and equilibrium binding techniques. The initial studies will utilize single bilayer vesicles and then progress to well-characterized model and native LPs. Since lifestyles determine, in part, the fatty acid composition of lipids, and the quantity and structure of the LPs that contain them, our studies should aid in our understanding of the factors that affect plasma LP turnover and lipid metabolism in general.
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