The applicant is ABIM certified in Internal Medicine (1986) and Cardiovascular Diseases (1989). This proposal represents training necessary for his development as an independent investigator in molecular cardiology. Techniques of protein purification gel electrophoresis, equilibrium enzymes assays, liposome preparation and general quantitative methodology as well as molecular biologic techniques of immunoblotting, DNA and RNA purifications and sequencing, will be used to develop an understanding of the mechanisms involved in regulation of intestinal cholesterol absorption. We have purified to homogeneity human pancreatic cholesterol esterase and triglyceride lipase and have shown that substrate cleavage occurs when these enzymes bind to the brush border membrane in a receptor-like manner at a heparin specific site . We have thus defined a mechanism for lipid absorption and defined a physiologic role for intestinal heparin. In the planned work we will isolate, purify and characterize the human brush border pancreatic cholesterol esterase receptor from CaCo-2 cells. This will be done with affinity chromatography with a synthesized cholesterol esterase-sepharose resin and elution with sequential salt and/or detergent containing gradients. This work will provide a matrix for detailed structure function analysis to evaluate the regulation of ligand receptor interaction. Using derived amino acid sequence data, we will clone and sequence its cDNA from a human intestinal cell library. This will serve as a basis to isolate the human genomic DNA and allow us to investigate genetic and biochemical regulatory mechanisms relating intestinal lipid absorption and atherogenesis. The applicant will work under the supervision of Dr. Louis Lange, an acknowledged leader in the enzymology and biochemistry of neutral lipids, who provides a strong role model both in basic science and clinical disciplines. His laboratory is fully equipped for the described experimental work and also provides a challenging and supportive intellectual environment with ties to both the Pharmacology, Pathology, and Metabolism Departments at Washington University. The applicant will work within the Division of Cardiology with 80 to 90% of his time devoted to basic research and 10 to 20% to clinical work throughout the award period.
