Identification of lipoxygenase expressing ω-6 dioxygenase activity by means of liquid chromatography


Authors: Peter Hoffman ;  Mária Cupáková 1;  Drahomíra Rauová 1;  Renáta Kollárová ;  Mária Pekárová ;  Marek Obložinský ;  Lýdia Bezáková
Authors‘ workplace: Katedra bunkovej a molekulárnej biológie liečiv, Farmaceutická fakulta Univerzita Komenského ;  Toxikologické a antidopingové centrum, Farmaceutická fakulta, Univerzita Komenského v Bratislave, Slovenská republika 1
Published in: Čes. slov. Farm., 2012; 61, 53-59
Category: Original Articles

Overview

Lipoxygenases (LOX) represent a family of lipid peroxidising enzymes which catalyse the reaction of achiral polyunsaturated fatty acids by oxygen forming chiral peroxide products possessing high positional stereospecific purity. The four double bonds of arachidonic acid, the main substrate of animal LOX, present the position for a wide range of enzymatic modifications enabling eicosanoid creation, unique molecules with biological significance. In this study, lipoxygenase from rat lung cytoplasma was isolated and purified to 40-fold by combining hydrophobic and gel chromatography. The forming positional specific fatty acid hydroxyl-isomers were separated on a non-polar system (RP-HPLC) and identified on a polar adsorbent (SP-HPLC). In the purified enzyme, dual positional specificity was demonstrated by the production of 12- and 15-HETE in the ratio of 1,0 : 1,38, which responds to the product spectrum of mammalian 15-LOX-1.

Keywords:
lipoxygenase • purification, HPLC analysis • 15-LOX-1


Sources

1. Werz O., Steinhilber, D. Therapeutic options for 5-lipoxygenase inhibitors. Pharmacol Therapeut. 2006; 112, 701–718.

2. Radmark O., Werz O., Steinhilber D., Samuelsson B. 5‑lipoxygenase: regulation of expression and enzyme activity. Trends Biochem Sci 2007; 32, 332–341.

3. González-Périz A., Claria, J. New approaches to the modulation of the cyclooxygenase-2 and 5-lipoxygenase pathways. Curr Top In Med Chem 2007; 7, 297–309.

4. Cyrus T., Pratico D., Zhao L., Witzlum J. L., Rader D. J., Rokach J., FitzGerald G. A., Funk C.D. Absence of 12/15-lipoxygenase expression decreases lipid peroxidation and atherogenesis in apolipoprotein E-deficient mice. Circulation 2001; 103, 2277–2282.

5. Iversen L., Kragballe K. Arachidonic acid metabolism in skin health and disease. Prostag Oth Lipid M 2000; 63, 25–42.

6. Powell W.S., Rokach J. Biochemistry, biology and chemistry of the 5-lipoxygenase product 5-oxo-ETE. Prog Lipid Res 2005; 44, 154–183.

7. Buczynski M. W., Dumlao D. S., Dennis E. A. Thematic review series: Proteomics. An integrated omics analysis of eicosanoid biology. J Lipid Res 2009; 50, 1015–1038.

8. Bradford, M. M. A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding. Anal Biochem 1979; 72, 248–254.

9. Flurkey W. H., Young L. W., Jen J. J. Separation of soybean lipoxygenase and peroxidase by hydrophobic chromatography. J Agric Food Chem 1978; 26, 1474–1476.

10. Hover C. G., Kulkarni A. P. A simple and efficient method for hemoglobin removal from mammalian tissue cytosol by zinc sulfate and its application to the study of lipoxygenase. Prostag Leukotr Ess 2000; 62, 97–105.

11. Calandria J. M., Marcheselli V. L., Mukherjee P. K., Uddin J., Winkler J. W., Petasis N. A., Bazan N. G. Selective survival rescue in 15-lipoxygenase-1-deficient retinal pigment epithelial cells by the novel docosahexaenoic acid-derived mediator, neuroprotectin D1. J Biol Chem 2009; 284, 17877–17882.

12. Jiang W. G., Watkins G., Douglas–Jones A., Mansel R. E. Reduction of isoforms of 15-lipoxygenase (15-LOX)-1 and 15-LOX-2 in human breast cancer. Prostag Leukotr Ess 2006; 74, 235–245.

13. Shureiqi I., Jiang W., Zuo X., Wu Y., Stimmel J. B., Leesnitzer L. M., Morris J. S., Fan H. Z., Fischer S. M., Lippman S. M. The 15-lipoxygenase-1 product 13-S-hydroxyoctadecadienoic acid down-regulates PPAR-∂ to induce apoptosis in colorectal cancer cells. P Natl Acad Sci USA 2003; 100, 9968–9973.

14. Liu C., Xu D., Liu L., Schain F., Brunnström A., Björkholm M., Claesson H. E., Sjöberg J. 15-lipoxygenase-1 induces expression and release of chemokines in cultured human lung epithelial cells. Am J Physiol 2009; 297, L196–L203.

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Pharmacy Clinical pharmacology
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