Assessment of 5-Lipoxygenase Activity and Cellular Distribution

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.