The purpose of the present proposal is to continue studies on the regulation of acylcoenzyme A:cholesterol acyltransferase (ACAT) and the role of this enzyme in the uptake and transport of cholesterol in the small intestine. The second part of the project will be devoted to the purification of hepatic ACAT. CaCo-2 cells, a newly established intestinal cell line, will be used to study the regulation of ACAT activity by micellar cholesterol, lipoprotein production, and membrane fatty acid modification. CaCo-2 cells will be incubated with bile salt micelles of various compositions containing cholesterol. The absorption of cholesterol from these micelles as well as its effect on ACAT activity will be determined. Experiments to assess the contribution of the absorbed cholesterol to the ACAT substrate pool will be performed to explain a possible mechanism for the regulation. CaCo-2 cells will be driven to produce lipoproteins by incubating them with high concentrations of fatty acids. The rate of lipoprotein synthesis and the effect on ACAT activity will be measured. Membrane fatty acid modification will be accomplished by culturing the cells in various fatty acids. After specified intervals, ACAT activity and membrane fatty acid content will be determined. In another series of experiments, ACAT activity will be modulated to assess the regulation of cholesterol absorption and lipoprotein synthesis in CaCo-2 cells by ACAT. In part two, rat liver will be used as starting material for ACAT purification, with eventual employment of human liver and cell lines. Various detergents in increasing concentrations will be tried initially to test their suitability for solubilization, ease of removal, and sensitivity of enzyme analysis. General chromatographic approaches such as gel filtration, ion exchange, hydrophobic, dye, hydroxylapatite, heparin-agarose, and lectin- agarose chromatography will be followed by gel affinity techniques. Analysis of ACAT protein will be done by polyacrylamide gel electrophoresis and gel filtration. Plans have been developed for the preparation of monoclonal antibodies and the use of these antibodies for the study of ACAT regulation in cells. The successful completion of both projects will greatly enhance our knowledge of intracellular cholesterol esterification and allow for further expansion and progression in this field.
