Glucuronidation is an important pathway in the biotransformation and excretion of exogenous drugs and endogenous compounds, such as bilirubin, steroids, bile and fatty acids, and thyroid hormones. The conjugation with glucuronic acid generally results in the increased solubility and decreased biological activity of a compound. However, glucuronidation has been shown to be involved in the bioactivation of compounds to mutagens and carcinogens or to pharmacologically active forms. UDP- glucuronosyltransferases (UGTs) are the family of enzymes responsible for this glucuronidation, which occurs predominantly in the liver. Since the liver is an epithelium which actively absorbs numerous substances from the blood, metabolizes them and secretes them into bile or urine, transport is an integral part of detoxification, a fact that often receives less attention that the biochemical transformations mentioned above. UDP-GlcUA, the essential co-substrate for all UGTs, is synthesized from UDP-Glc by UDP-glucose dehydrogenase in the cytosol of the hepatocyte. Considering the putative lumenal location of the active site for UGTs, a mechanism for the translocation of UDP-GlcUA from the cytosol to the ER lumen is required. The hepatic ER transport of UDP-GlcUA has been partially characterized on the functional level by us and other researchers; however, no information is available concerning the enzyme(s) responsible for the translocation. Our preliminary results, from studies combining uptake assays, photoaffinity labeling and the use of inhibitors, have identified two putative protein candidates as the UDP-GlcUA transporter in rat liver membranes: an 80 kDa protein and/or the UGTs themselves. The goal of this proposal is to identify, purify and characterize the protein(s) involved in the transport of UDP-GlcUA in order to better characterize the role of UDP-GlcUA transport in detoxification and glucuronidation activities.
The specific aims of this proposal are: 1. Purification and reconstitution of UDP-GlcUA transporter protein(s). The putative 80 kDa UDP-GlcUA transporter and UGT isoenzymes will be purified from isolated rat liver ER membranes by conventional solubilization and chromatographic techniques. Purified protein will be reconstituted and UDP-GlcUA uptake will be determined by rapid filtration techniques. 2. Structural characterization of the transporter(s). Preparative SDS-PAGE purification of the 80 kDa protein and UGTs photolabeled with [32P]5- azido-UDP-GlcUA will be done to isolate these proteins in sequencable amounts. These purified proteins will be used to generate polyclonal antibodies and for partial protein synthesis and active-site amino acid analysis. The elucidation of the mechanisms responsible for the microsomal membrane translocation of UDP-GlcUA will provide basic information that may impact on the role of glucuronidation in the safety and metabolism of drugs used in humans.
