9723771 Cuebas The biosynthesis of bile acids from cholesterol is an important process that occurs in liver cells of all vertebrates. The objective of this research is to characterize the enzyme system(s) responsible for the(-oxidative modifications of the steroid side chain in the formation of bile acids in several lower vertebrate species and in the rat. Since the end-products of cholesterol degradation are bile acids, this project will improve our understanding of the processes by which cholesterol is eliminated from organisms. There is a lack of information regarding the enzymes in peroxisomes that catalyze these conversions and the stereochemical mechanisms by which these enzyme reactions proceed. This project will involve the chemical synthesis of the Coenzyme A derivatives of putative sterol intermediates during bile acid biosynthesis as well as the purification and characterization of the enzymes found to catalyze these transformations. Spectrophotometry, HPLC, NMR, and GC/MS will be used to determine the kinetic constants, substrate specificities, and the stereochemistries of these reactions. Enzymes will be purified from liver tissue using conventional chromatography and FPLC. Since peroxisomes harbor two different multifunctional proteins that catalyze the formation of (-hydroxyacyl-CoA intermediates with opposite stereochemistries, it will be of significance to determine which of these multifunctional proteins is necessary and/or sufficient for bile acid biosynthesis. This biosynthetic pathway will be studied in liver obtained from toad, bullfrog, lizard, alligator, and the rat. Since the extent of side chain modification in primary bile acids appears to be correlated to the degree of evolutionary development of the particular class of vertebrate, these studies will uncover species differences in(-oxidative enzyme systems responsible for bile acid biosynthesis and thereby provide valuable information in tracing evolutionary relationships between species. To aid in the detection o f auxiliary bile acid biosynthetic pathways, site-directed irreversible inhibitors will be synthesized that specifically inhibit enzymes catalyzing the peroxisomal oxidase and thiolase steps. Results from this project will not only make a significant contribution to our present knowledge of bile acid biosynthesis in mammals, but they will also uncover the enzymology behind the evolutionary diversity of bile acids produced by various species of vertebrates throughout the animal kingdom. The conversion of cholesterol to bile acids is an important process that occurs in liver cells of all vertebrates. The bile acids are detergents that are secreted into the digestive tract and are essential for the digestion of fatty materials that are consumed in the diet. In addition, the fecal excretion of bile acids is the primary mechanism by which organisms can eliminate cholesterol from their body. The objective of this project is to investigate the enzymatic mechanisms that have evolved for the conversion of cholesterol to bile acids in mammals, reptiles, and various amphibians and thereby provide valuable information concerning cholesterol metabolism in various species. Results from this project will not only make a significant contribution to our present knowledge of bile acid biosynthesis in mammals, but they will also uncover the details behind the evolutionary diversity of bile acids produced by various species of vertebrates throughout the animal kingdom.
