Hepatic glucuronidation represents a major metabolic pathway for the biotransformation of hydrophobic endobiotics, such as bilirubin and testosterone, into more water soluble compounds which may be excreted in bile or urine. This conjugation reaction also represents an important detoxification and excretory pathway for xenobiotics such as morphine, acetaminophen, phenols, and napthols. UDP-glucuronosyltransferase, the enzyme responsible for glucuronidation, is localized predominately on the cisternal membrane of the endoplasmic reticulum. This orientation of the enzyme demands that the hydrophilic UDP-glucuronic acid (UDPGA) substrate, which acts as the essential donor of the glucuronide moiety and is synthesized in the hepatocyte cytosol, be translocated across the endoplasmic reticulum membrane to its active site on the enzyme. The primary goal of this research proposal is the identification and characterization of the mechanism(s) responsible for the transport of this important metabolic co-substrate across the hydrophobic endoplasmic reticulum bilayer. Thorough characterization of this process will result from the systemic application of refined kinetic assays, photoaffinity labelling and affinity chromatography, and expression cloning of the putative UDP-glucuronic acid transport protein in Xenopus laevis oocytes. These investigations will permit description of the microsomal UDP-glucuronic acid transporter at the amino acid and cDNA levels. With this information, insight into the role which UDP glucuronic acid transport plays in states of normal and impaired glucuronidation may be obtained. Completion of the description of this transport process in normal animal models will be followed by analysis of microsomal UDP-glucuronic acid translocation in mutant animal models (e.g. Gunn rats) and animal models in which physiologic states may mimic human conditions (e.g. neonatal and fasting states). Finally, this transport process will be examined in human explanted livers to assess its role in human states of health and disease (e.g. Crigler-Najjar syndromes). The knowledge obtained from these studies regarding the modulation and regulation of UDP-glucuronic acid transport may provide new insight into a host of metabolic processes including, but not limited to, hormone homeostasis, pharmacokinetics and xenobiotic detoxification.
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