Leukotrienes (LT) are a family of biologically active compounds which are synthesized via the 5-lipoxygenase pathway. LTC4 is formed by the conjugation of glutathione to LTA4, a labile epoxide intermediate. LTC4 can be sequentially converted to LTD4 and LTE4 by the removal of glutamate and glycine, respectively. LTC4 and LTD4 are very potent constrictor of bronchioles and arterioles and cause plasma exudation. We have been successful to generate a cell-free leukotriene enzyme system from rat basophilic leukemia (RBL-1) cells which we utilized to characterize leukotriene biosynthesis. Now we propose to use the RBL-1 cell system to characterize in detail the glutathione transferase responsible for LTC4 formation. This enzyme appears to be quite different from other GSH transferases with regard to subcellular localization, substrate specificity and function. We will continue to the study the subcellular localization of this enzyme. Circumstantial evidence suggests that it may be in the granules. Therefore we will attempt to obtain a relatively pure granule preparation and also confirm the localization by raising specific antibodies. We will also probe into the possible role of LTC4 in the granule release reaction by inhibiting the conversion of LTC4 to LTD4 and by glutathione enrichment which enhances LTC4 synthesis. We propose to purify the enzyme and study it in comparison with other glutathione transferases. We propose to characterize the enzyme by (1) cross reactivity studies with antibodies to various soluble glutathione transferase, (2) establishing its inhibition profile as compared to soluble and microsomal glutathione transferases, (3) determining its substrate specificity and (4) probing which functional groups are important at the active site. We also plan to determine certain physical characteristics of this enzyme, such as apparent molecular weight and isoelectric point and compare peptide maps obtained from this enzyme with the peptide maps of other glutathione transferases. We hope to definitely demonstrate the difference of the LTC4-synthesizing enzyme from other glutathione transferases which would make it possible to design selective inhibitors of LTC4 biosynthesis.
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