Prostaglandins and the related eicosanoids are a family of biologically potent fatty acids derived from arachidonic acid. These substances are very short-lived in vivo and are rapidly inactivated by first oxidation of the 15(s) -hydroxyl group catalyzed by NAD+ -dependent 15- hydroxyprostaglandin dehydrogenase (15-PGDH) followed by reduction of 13.14-double bond catalyzed by 15-ketoprosta-glandin delta13 -reductase (13-PGR). Cloning, characterization and structure and activity relationship of 15-PGDH have been extensively studied. Recently we have cloned the genomic DNA of mouse 15-PGDH gene and have sequenced the 5'-flanking region of the gene. The structural information allows us to pursue the following specific aims. Firstly, we propose to demonstrate promoter activity. Secondly, we plan to characterize the promoter region by deletion analysis and site-directed mutagenesis and to identify specific regulatory elements involved in the gene expression initiated by phorbol ester, steroid hormones, v-src and I1-6. Thirdly, we propose to localize the 15-PGDH gene in chromosomes. With respect to 13-PGR, we propose to clone and express the cDNA from porcine lung followed by characterization of the enzyme with respect to substrate and coenzyme specificity. Further characterization includes identification of the binding domains of NSDH and 15-ketoprostaglandins by photoaffinity labeling studies and elucidation of the structure and activity relationship by site- directed mutagenesis. The results of this research program should provide a molecular basis of our understanding of the regulatory mechanisms and the structural and function of two key catabolic enzymes of prostaglandins.
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