Glucuronidation of therapeutically useful drugs and endobiotics such as steroids and bilirubin regulates the duration of action and toxicity of these substances, especially in humans. A family of microsomal UDP- glucuronosyltransferases (UDPGTs) catalyze these reactions and, in certain cases, unique UDPGTs exist in human liver. The objective of this proposal is to reveal the functional and structural properties of UDPGTs mediating the 3-0 and 6-0-glucuronidation of morphine, tertiary amines other than morphine (e.g., tripelennamine, amitryptyline). Specifically, morphine UDPGTs will be purified to homogeneity from rat and human liver microsomes and compared with respect to their substrate specificity, NH2-terminal amino acid sequences and active site peptide amino acid sequences. cDNA cloning of rat and human liver UDPGTs will be performed using polymerase chain reaction techniques with oligonucleotides whose synthesis will be based on amino acid sequence knowledge. Specific active site photoaffinity labeling using [3H]-flunitrazepam (FNZ) will be useful in accomplishing a number of these aims. Active and FNZ-labeled morphine UDPGT will be purified to homogeneity using Fractogel, chromatofocusing and affinity chromatography. Human liver tertiary amine UDPGT will also be studied using photoaffinity labeling, selected antibody immunorecognition, chromatographic separations, NH2-terminal amino acid sequencing and cDNA cloning techniques. Gender-related or drug-induced increases in human liver estriol UDPGT will also be studied. This research will provide information which will allow for predictions of potential drug-drug interaction, methods for study or possible human polymorphisms of UDPGTs, and an understanding of the regulation of metabolism by glucuronidation.
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