The principal focus is the elucidation of the roles of lipoxygenase products of arachidonic acid, such as leukotrienes, in the mediation and modulation of human hypersensitivity reactions. The major objectives are the definition of the enzymatic properties of lipoxygenases and epoxide hydrolases of murine mast cells and human leukocytes and the characterization of the modlecular bases for cellular expression and specificity of leukocyte receptors for leukotrienes. The mechanisms of generation and recognition of leukotrienes by leukocytes are linked by their involvement in membrane integrity and activation of cellular functions of leukocytes that serve as the source of the mediators. The time-course and stimulus-specificity of the development of lipoxygenases and leukotriene receptors will be examined in cultured lines of human leukocytes that are induced to differentiate in vitro. Lipoxygenases and epoxide hydrolases will be purified from fully differentiated mast cells and leukocytes for determination of amino acid sequences and preparation of antibodies, in order to study subcellular distribution and the genetic determinants of expression of enzymatic activities. Extensive investigations of the enzymology of lipoxygenation and epoxide hydration will include the characterization of pharmacological inhibitors and of the feed-back regulatory effects of products, as well as the mechanisms of double lipoxygenation reactions in cells containing two or more lipoxygenases capable of concerted action. Leukotriene B4 receptors on differentiated leukocytes will be photo-affinity labeled and affinity cross-linked with radioactive ligands for quantitative purification and assessment of physical properties. Purified intact leukotriene B4 receptors and affinity-labeled substituent peptides will be utilized for determination of amino acid sequences and preparation of antibodies. Antibodies to leukotriene receptors of sufficient affinity will be utilized to establish the characteristics of subcellular distribution, processing, and regeneration or recycling of the receptors in unsimulated, stimulated, and chemotactically deactivated leukocytes. Amino acid sequences of leukotriene receptor proteins and antibodies to the receptors will be used to analyze the molecular biology of generation, specificity, and cellular distribution of the leukocyte receptors for leukotriene B4. Biochemical and immunochemical methods will be applied to the evaluation of the receptor-associated proteins and cellular events critical for the coupling of receptor stimulation to leukocyte activation.
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