Abstract
This chapter discusses the binding assay in intact cells, membranes, and a detergent-solubilized preparation and a facile neutrophil aggregation assay that can be used to correlate binding data with biological information. The leukotrienes are metabolites of arachidonic acid whose potent biological activities suggest an important role in proinflammatory and hypersensitivity reactions. The initial step in the biosynthesis of leukotrienes is catalyzed by the enzyme 5-1ipoxygenase, which peroxidizes arachidonic acid at carbon-5 to form 5(S)-hydroperoxy-6-trans-8,11,14.cis-icosatetraenoic acid (5- HPETE). The hydroperoxide compound is then converted to the unstable allylic epoxide intermediate, leukotriene A4 by the enzyme leukotriene A4 synthase. Leukotriene B4 is a potent mediator of polymorphonuclear leukocytes (PMN) aggregation, chemotaxis, superoxide generation, and lysosomal enzyme release. The chapter also discusses the purification of human PMN. A convenient, readily accessible source of material that can be used to measure leukotriene B4 binding as well as a bioassay for leukotriene B4 is purified human PMN. The PMN are isolated from whole human blood by a combination of Dextran sedimentation and Ficoll–Hypaque centrifugation.
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