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12-Lipoxygenases

Rapp, Johanna
http://rave.ohiolink.edu/etdc/view?acc_num=toledo1164900972

Abstract Details

2006, Master of Science, University of Toledo, College of Arts and Sciences.
The lipoxygenase enzymes are non-heme, non-sulfur iron dioxygenases that are widely distributed throughout plants, animals, fungi and some bacteria. This family of enzymes plays a major role in polyunsaturated fatty acid metabolism because they catalyze the incorporation of molecular oxygen into fatty acids containing a 1,4-pentadiene moiety producing hydroperoxide products. In mammals, the products of the dioxygenation catalyzed by lipoxygenases, hydroperoxyeicosatetraenoic acid (HPETEs), are intermediates in the formation of bioregulators including leukotrienes, hepoxilins and HETEs, that are implicated in a variety of human conditions such as inflammation, fever, arthritis and cancer. 12-Lipoxygenases are mammalian lipoxygenases that catalyze the conversion of arachidonic acid into 12-hydroperoxy-5,8,10,14-eicosatetraenoic acid (12-HPETE). Actually, 12-HPETE is a precursor to the formation of the hydroxy fatty acid 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) and also epoxy fatty acids called hepoxylins. There are three isoforms of 12-lipoxygenase named after the cells where they are found: leukocyte, platelet and epidermis type-enzymes. Even if the leukocyte and the platelet 12-lipoxygenase catalyze the same reaction, they differ by their tissue distribution, their substrate specificity, their primary structure and size. They are also involved in different kinds of diseases as inflammation, hypertension and diabetes. The goal of this research was to investigate the human platelet and porcine leukocyte 12-lipoxygenases. In order to study both 12-lipoxygenases, they need to be expressed, purified and in the best case crystallized to get the 3D structure. This thesis presents the expression and purification protocols that lead to the preparation of large amounts of pure porcine leukocyte 12-lipoxygenase. Several biophysical analyses were performed to characterize the protein in solution such as dynamic light scattering (DLS), differential scanning calorimetry (DSC), EPR spectroscopy and mass spectrometry. Many crystallization experiments were also tried in order to obtain diffraction-quality crystals of porcine leukocyte 12-lipoxygenase. As for the human platelet 12-lipoxygenase, the expression step turned out to be less successful, and leads to low yields and unsatisfactory purity.
Max O. Funk (Advisor)
105 p.
Chemistry, Biochemistry
Lipoxygenase; Expression in E.coli; Crystallization; EPR; DSC; DLS; Mass spectrometry; HPETE

Recommended Citations

Citations

  • Rapp, J. (2006). 12-Lipoxygenases [Master's thesis, University of Toledo]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1164900972

    APA Style (7th edition)

  • Rapp, Johanna. 12-Lipoxygenases. 2006. University of Toledo, Master's thesis. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=toledo1164900972.

    MLA Style (8th edition)

  • Rapp, Johanna. "12-Lipoxygenases." Master's thesis, University of Toledo, 2006. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1164900972

    Chicago Manual of Style (17th edition)

toledo1164900972
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© 2006, all rights reserved.
This open access ETD is published by University of Toledo and OhioLINK.