The long-term goal of the proposed research is a better understanding of the mechanism and functions of bile acid glucuronidation. Glucuronides of the quantitatively predominant bile acids occur in small amounts in the healthy organism and become prominent during cholestasis. In this situation, glucuronide formation helps to detoxify and remove the excess of bile acids that would otherwise accumulate to toxic levels. However, for a particular subclass of bile acids, the short-chain bile acids, glucuronidation appears to be the predominant or only mode of conjugation, even in the healthy state. Moreover, this class of bile acids gives rise not only to the known hydroxyl-linked glucuronides, but, in a strictly substrate-specific way, to other positional isomers, namely the carboxyl-linked glucuronides and the diglucuronides. The recent rise of interest in short-chain bile acids reflects the promise these compounds hold for a better understanding of the metabolism of steroidal hormones, of the interplay of the metabolism of intestinal bacteria with that of the liver, and other questions of hepatic biochemistry. The present proposal deals with the enzymology of the carboxyl-directed glucuronidation of bile acids. The UDP-glucuronyltransferase responsible for this reaction will be purified to homogeneity using a combination of chromatofocusing and affinity chromatography on immobilized UDP, both techniques that have proven valuable for related glucuronyltransferases. If needed, affinity chromatography on short-chain bile acids immobilized via the steroidal nucleus will be added. The enzyme will be characterized and compared with other UDP-glucuronyltransferases in terms of physical characteristics, substrate-specificity and competition, ontogenic behavior, inducibility by drugs, and immunological cross-reactivity. Specific inhibitors of the enzyme will be sought; in particular, a novel method of photoaffinity labeling designed to minimize background labeling will be applied to the enzyme. Finally, the carboxyl-specific bile acid UDP-glucuronyltransferase will be reconstituted and questions relating to enzyme latency and substrate transport will be examined using this system.
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