Research Project
3503459
Lipoxygenases constitute a family of non-heme iron-containing dioxygenases that have been demonstrated to produce a wide range of physiologically active metabolites. The enzyme 12-lipoxygenase (12-LX) is present in platelets, leukocytes, lung and brain. 12-LX converts arachidonic acid to 12-HPETE, which can then be converted to 12-HETE. More recently, epoxy (hepoxylins A(3) and B(3)) dihydroxy and trihydroxy derivatives of 12(S)- HPETE have been described. The enzyme 5-lipoxygenase (5-LX) plays a pivotal role in the biosynthesis of leukotrienes, which are important in the regulation of numerous biological processes, ranging from acute inflammation and thrombosis to chronic cardiovascular disorders such as atherosclerosis. The enzyme 15-lipoxygenase (15-LX) peroxidizes arachidonic acid to form 15(S)-HPETE, which can be converted to 15(S)-HETE and the trihydroxy compounds lipoxin A(4) and lipoxin B(4), which exhibit a range of biological activities. Commercial availability of purified preparations of human lipoxygenases and a complete set of molecular probes (nucleic acid probes as well as poly- and monoclonal antibodies) will greatly facilitate our understanding of the physiologic and pathophysiologic roles of these proteins. The ultimate goal of this project is the development of commercially viable methods for purification to homogeneity of human lipoxygenases, the production of polyclonal and monoclonal antibodies, as well as nucleic acid probes specific for each of these enzymes. These reagents will then be marketed to scientists investigating the distribution and regulation of human 5-, 12- and 15-lipoxygenase enzymes in normal and diseased individuals. In Phase I, we propose to further develop methods to stabilize and purify human platelet 12-LX suitable for commercial production, and will also generate rabbit polyclonal antibodies specific for this protein. In subsequent Phase II, we plan to: (a) generate monoclonal antibodies specific for platelet 12-LX, (b) purify human 5-LX, 15-LX and leukocyte 12-LX and generate polyclonal and monoclonal antibodies specific for each of these enzymes, (c) synthesize additional nucleic acid probes, as needed, to provide a complete set of probes for human lipoxygenases, and (d) screen established human cell lines in order to identify convenience standards for assays of expression and activity of each of these enzymes.
