Prostaglandins and the related eicosanoids are a family of biologically potent fatty acids derived from arachidonic acid. These potent products are very short lived in vivo and are rapidly inactivated to 15-keto derivatives catalyzed by NAD+ -dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH). Therefore, tissue and circulating levels eicosanoids are regulated not only by their synthesis but also by their metabolism. We have purified 15-PGDH from human placenta to apparent homogeneity and produced both polyclonal and monoclonal antibodies against the enzyme. We have also cloned and sequenced a full length cDNA using these antibodies and some partial amino acid sequences of the enzyme. The availability of these biochemical tools allows us to pursue four specific objectives concerning the structure and function of this enzyme. Firstly, we propose to analyze the structure-activity relationship of 15-PGDH by identifying the prostaglandin and NAD+ binding sites using photoaffinity labeling techniques and site directed mutagenesis of 15-PGDH cDNA at the predicted regions. Secondly, we propose to clone and sequence the 5'-flanking region of 15-PGDH gene and identify regulatory regions for future studies on the regulation of 15-PGDH gene expression. Thirdly, we propose to elucidate the role of 15-PGDH in prostaglandin metabolism and function by studying the effects of prostaglandins on mitogenic responses, adipose cell differentiation and osteoblast differentiation in cells expressing 15-PGDH activity. Finally, we plan to elucidate the molecular basis of defective prostaglandin metabolism in genetic hypertensive rats. The results of this program should aid in understanding the biochemistry, physiology and pathophysiology of the enzyme.
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