The long-term goal of this research is to understand how the distribution of free and esterified cholesterol is regulated, both among tissue and among intracellular pools. This project is concerned with specific regulatory aspects of the overall process. Primary focus is on the regulation of the interfacial reaction. CHOLESTERYL ESTER + WATER EQUILIBRIUM WITH FATTY ACID + CHOLESTEROL as catalyzed by mammalian cholesterol esterases. Related to this is the regulation of the spontaneous transport of lipid substrate and products to and away from the reaction site. In this regard, the partitioning of free cholesterol into the aqueous phase and the movement of cholesterol and cholesteryl ester across bilayers will be addressed. An important characteristic of both the hydrolysis reaction and the transport reactions is that they are interfacial. Hence, the major experimental emphasis is on the role of lipid- lipid and lipid-protein interactions in surface phases in the regulation of these processes. Specifically, rates and extents of enzyme adsorption to and substrate hydrolysis in surface phases at the argon-buffer interface will be measured. The enzymes to be used are pancreatic carboxylester lipase, lysosomal cholesterol esterase and hormone sensitive cholesterol esterase. Emphasis is on determining how these enzymes are regulated by specific lipid- lipid interactions. From the results obtained apparent enzyme specificity can be separated into physical and chemical specificities. Studies of the regulation of the spontaneous transfer of cholesterol and cholesteryl ester across bilayers will reveal under what conditions cholesterol esterases may facilitate cholesterol movement across bilayers. The achievement of these aims will greatly increase our understanding of the regulation of the distribution of cholesterol between chemical forms and physical pools in vivo.
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