The purpose of this application is to investigate interactions between cholesterol and proteins in the human erythrocyte membrane and other mammalian membrane systems, and in the human serum lipoproteins, low density kipoprotein (LDL) and high density lipoprotein (HDL). This investigation is anticipated to provide new ideas concerning the role of cholesterol in the pathogenesis of atherosclerosis and in normal mammalian cell growth. Specifically, cholesterol-protein interactions may modulate the activity of the plasma membrane Na+K+ATPase, thereby biochemically linking human obesity, high serum cholesterol levels and atherosclerosis; cholesterol-protein interactions are likely different in human LDL than in human HDL, which may relate to the opposite roles these two lipoproteins play in the pathogenesis of atherosclerosis; cholesterol-protein interactions may provide a biochemical basis for the preferential location of cholesterol in the plasma membrane of mammalian cells. Cholesterol-protein interactions will be studied in the human erythrocyte membrane and for comparison, interactions with the Na+K+ATPase will be studied in the erythrocyte membranes and in recombinants of enzyme from kidney medulla. Cholesteral-protein interactions will also be studied in LDL and HDL. In addtiion to the basic biochemistry devoted to carefully preparing and characterizing these systems, six approaches to studying cholesterol-protein interactions will be employed, all but one of them new in application to this question. 31P nuclear magnetic resonance (NMR) of the phospholipids and 13C NMR of 13C labelled cholesterol will be used, as will circular dichroism (CD) and fluorescence of a derivative of cholesterol, CD and fluorescence of parinaric acid, a lipid probe, phase transition behavior of phospholipids, and enzyme activity of the ATPases. This multifaceted approach is expected to provide a rather complete picture of cholesterol behavior with respect to protein, in a field where almost no information was available previously.
