The specific aims of the application are 1) to synthesize and purify peptide fragments of apolipoprotein E in order to determine the receptor and lipid-binding regions of the protein, 2) to synthesize apoE peptides with amino acid substitutions to determine the importance of specific amino acids to receptor binding and 3) to model the apoE receptor binding region using a model lipid associating peptide previously shown to display many of the properties of the apolipoproteins. The peptides of interest will be synthesized by solid phase methodology and purified by ion exchange chromatography. The amino acid composition will be determined by amino acid analysis. The purity of the peptides will be determined of isoelectric focusing and reversed phase high performance liquid chromatography on radially compressed C18 silica solumns. The pruified peptides will be tested for lipid binding using CD spectroscopy and density gradient ultracentrifugation. The peptides will be mixed with cholesterol-rich dispersions and tested for hepatocyte receptor binding in competition with apoE containing dispersions. Elucidating the relationship between receptor binding and lipid binding will further our understanding of how cholesterol is taken up and metabolized by the liver. A knowlegde of the protein determinants for recptor binding could clarify the difference in hepatic uptake of various lipoprotein remnants in disease states and could probably lead to effective therapy for Type III hyperlipoproteinemia where a defect has been demonstrated in apolipoprotein E receptor binding due to a substitution of cysteine for arginine in the amino acid sequence.
