Lecithin cholesterol acyl transferase (LCAT) is the enzyme responsible for the extracellular esterification of cholesterol and for transformations of high density lipoproteins (HDL) in plasma. So far, some enzymatic properties of LCAT have been investigated using native lipoproteins or synthetic vesicles of phosphatidylcholines and cholesterol with added apolipoproteins. These substrates have several disadvantages. Unfractionated plasma or isolated lipoproteins are physiological substrates of the enzyme, but have variable and uncontrollable lipid and apolipoprotein compositions. Sonicated vesicles offer the advantage of a chemically defined system, but present the problems of stability in the presence of apolipoproteins, a limited capacity for cholesterol ester storage, and a major morphological difference from the natural substrates. We propose to use synthetic, discoidal or spheroidal complexes containing apolipoprotein A-I (apo A-I), phosphatidylcholines, and cholesterol, prepared by a cholate dialysis method developed in our laboratory. These chemically defined, stable analogs of nascent or mature HDL particles, will be used to investigate systematically the substrate specificity of purified LCAT. We plan to examine the acyl donor specificity, the effect of the physical state of the lipids, the effect of variable phosphatidylcholine to cholesterol ratios, the cofactor role of apo A-I, and the effect of the size and shape of substrate particles on the LCAT reaction. The properties of the substrate particles will be investigated in terms of their chemical composition, size (by gel-filtration, electron microscopy, analytical ultracentrifugation, and fluorescence polarization methods), spectral properties of the apolipoproteins (by fluorescence spectroscopy, fluorescence quenching, and CD measurements), and the physical state of the lipid domains (from fluorescence polarization of diphenylhexatriene as a function of temperature). The reactivity of the various substrates with LCAT will be determined from initial velocity measurements under conditions where the enzyme is saturated with substrate. Temperature dependence of initial reaction rates will yield Arrhenius plots and activation energies.
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