LONG-TERM OBJECTIVE: Elucidation of the roles hepatic lysosomal and cytosolic cholesteryl ester hydrolases (CEH) in cholesterol homeostasis and bile acid and lipoprotein metabolism. Studies are designed to 1) measure and characterize CEH enzymes in the different subcellular compartments of the rat liver, 2) identify factors and mechanisms involved in their regulation, 3) differentiate their properties and functions with regard to hydrolysis of cholesteryl esters from lipoproteins and intracellular stores and provision of substrate for bile acid synthesis and biliary cholesterol secretion and 4) determine effects of manipulation of these enzymes on cholesterol pools and bile acid and lipoprotein metabolism. Cytosolic and lysosomal CEH enzymes will be purified by HPLC methods, characterized by peptide analysis, amino acid sequencing and group specific probes, and used to produce polyclonal and monoclonal antibodies. Specific activities of CEH enzymes will be measured in subcellular fractions of freshly prepared or cultured hepatocytes from normal, cholesterol-fed or cholestyramine treated rats. Effects of CEH activators, inhibitors, bile acids, hormones lipoproteins and other potential effectors of cholesterol homeostasis on CEH activity will be determine. In addition, 1) other enzymes of cholesterol metabolism (HMG-CoA reductase, ACAT, cholesterol 7-alpha- hydroxylase), 2) lipoprotein uptake, 3) bile acid and cholesterol synthesis and secretion and 4) free and esterified cholesterol will be measured in order to establish correlations and clarify compensatory responses under various conditions. Effects of manipulations on bile acid synthesis and cholesterol secretion will be confirmed in rats with extracorporeal enterohepatic circulation. Various hepatic cholesterol pools will be differentially radiolabeled in order to differentiate the functions of the CEH enzymes. Specific antibodies will be used in enzyme linked immunoabsorbent (ELISA) and Western blot assays to measure levels of CEH protein in specific subcellular fractions of cultured hepatocytes, liver and other tissues in order to localize and quantitate CEH enzymes as a function of the various manipulations and to detect enzyme induction of covalent modification. Effects of protein kinase and phosphatase activities on purified enzymes will also be studied.
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