Abstract
5-Lipoxygenase (5-LO) catalyzes the conversion of arachidonic acid to leukotriene mediators of inflammation (1,2). Leukotriene biosynthesis in intact cells involves redistribution of 5-LO from a soluble cellular compartment where it is inactive to the nuclear envelope where its association with an activating protein, termed FLAP, is required for activation and optimal catalysis (3). The mechanism and extent of this redistribution and activation varies among different leukocytes and the choice of agonist (4). Although the enzymatic importance of 5-LO in leukotriene biosynthesis is well documented, nonenzymatic properties of 5-LO that govern its cellular distribution (4) and participation in signal transduction cascades (5–7) have only recently been investigated. This combination of enzymatic and nonenzymatic cellular biology makes 5-LO a complex macromolecule to study in terms of its cellular distribution and activation. The following series of protocols provides a means for determining the distribution/redistribution and activity profile of 5-LO in resting and activated human polymorphonuclear cells (8) and granulocytic HL-60 cells (9). The protocols that are presented provide methodology pertinent to cellular fractionation to generate a cytosolic and membrane fraction; cellular fractionation to generate cellular and nuclear soluble and membrane fractions; determination of 5-LO activity by reversed phase HPLC; and determination of 5-LO cellular distribution by Western analysis.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Samuelsson, B. (1983) Leukotrienes: mediators of immediate hypersensitivity reactions and inflammation. Science 220, 568–575.
Lewis, R. J., Austen, K. F., and Soberman, R. (1990) Leukotrienes and other products of the 5-lipoxygenase pathway. N. Engl. J. Med. 323, 645–655.
Brock, T. G., McNish, R. W., and Peters-Golden, M. (1995) Translocation and leukotriene synthetic capacity of nuclear 5-lipoxygenase in rat basophilic leukemia cells and alveolar macrophages. J. Biol. Chem. 270, 21,652–21,658.
Ford-Hutchinson, A. W., Gresser, M., and Young, R. N. (1994) 5-lipoxygenase. Ann. Rev. Biochem. 63, 383–417.
Lepley, R. A. and Fitzpatrick, F. A. (1994) 5-Lipoxygenase contains a functional Src homology 3-binding motif that interacts with the Src homology 3 domain of Grb2 and cytoskeletal proteins. J. Biol. Chem. 269, 24,163–24,168.
Lepley, R. A., Muskardin, D. T. and Fitzpatrick, F. A. (1996) Tyrosine kinase activity modulates catalysis and translocation of cellular 5-lipoxygenase. J. Biol. Chem. 271, 6179–6184.
Lepley, R. A. and Fitzpatrick, F. A. (1996) Inhibition of mitogen-activated protein kinase kinase blocks activation and redistribution of 5-lipoxygenase in HL-60 cells. Arch. Biochem. Biophys. 331, 141–144.
Young, S. K., Worthen, G. S., Haslett, C., Tonnesen, M. G., and Henson, P. M. (1990) Interaction between chemoattractants and bacterial lipopolysaccharide in the induction and enhancement of neutrophil adhesion. Am. J. Resp. Cell. Mol. Biol. 2, 523–532.
Collins, S. J., Gallo, R. C., and Gallagher, R. E. (1977) Continuous growth and differentiation of human myeloid leukaemic cells in suspension culture. Nature 270, 347–349.
Woods, J. W., Evans, J. F., Ethier, D., Scott, S., Vickers, P. J., Hearn, L., Heibein, J. A., Charleson, S., and Singer, I. I. (1993) 5-Lipoxygenase and 5-lipoxygenase-activating protein are localized in the nuclear envelope of activated human leukocytes. J. Exp. Med. 178, 1935–1946.
Towbin, H., Staehlin, T., and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 4350–4354.
Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 1999 Humana Press Inc.
About this protocol
Cite this protocol
Lepley, R.A. (1999). Assessment of 5-Lipoxygenase Activity and Cellular Distribution. In: Lianos, E.A. (eds) Eicosanoid Protocols. Methods in Molecular Biology, vol 120. Humana Press. https://doi.org/10.1385/1-59259-263-5:201
Download citation
DOI: https://doi.org/10.1385/1-59259-263-5:201
Publisher Name: Humana Press
Print ISBN: 978-0-89603-667-3
Online ISBN: 978-1-59259-263-0
eBook Packages: Springer Protocols