Abstract
UDP-glucose dehydrogenase (Ugd; EC 1.1.1.22) catalyzes the oxidation of UDP-glucose to UDP-glucuronic acid accompanied with the reduction of 2 moles of NAD. In liver, UDP-GlcA reacts with xenobiotic and endobiotic compounds to aid in their solubilization and excretion. The complete human ugd gene was isolated from phage and BAC library by using the previously isolated Ugd cDNA fragments as the probes. The ugd gene was found to be composed of 12 exons, with a size more than 25-kb in length. Fluorescence in situ hybridization has assigned the gene to chromosome 4p13. In comparison with the mouse gene, the exon-intron splice junctions in human ugd gene are similar to that of the mouse gene except that 2 additional exons were found in the human gene. The transcriptional start site of ugd was determined by primer extension and farther confirmed by RT-PCR, indicating that the start sites are located at nucleotide positions -358 and from -347 to -344 region upstream of ATG. An expression construct has been generated that allows the production of soluble and functional GST-Ugd fusion protein in E. coli Topp 2 cells. An antibody specifically recognized the human Ugd was generated and used to study the tissue and subcellular location of the protein. The Ugd was found to be present in a variety of tissue sites including lung, kidney, heart, liver and skeletal muscle. The immunofluorescence assay showed that the Ugd was localized in the HepG2 cytosol and possibly distributed more concentrated surrounding the ER and Golgi. The uneven distribution consistent with the cell fractionation results indicated that the membrane fractions contained the most of the Ugd.