Abstract
Leukotriene A(4) (LTA(4)) is a chemically unstable triene epoxide product of 5-lipoxygenase metabolism of arachidonic acid. Despite this chemical reactivity and its synthesis at the perinuclear membrane, LTA(4) is enzymatically converted into the cysteinyl leukotrienes and leukotriene B(4). Furthermore, LTA(4) participates in transcellular biosynthesis and is thus transferred between cells as an intact molecule. A cytosolic fatty acid-binding protein present in the rat basophilic leukemia cells was identified using mass spectrometry. This protein was determined to be the stabilizing factor present in the cell cytosol responsible for increasing the effective chemical half-life of LTA(4). Rat epithelial fatty acid-binding protein (E-FABP) was isolated using partial protein purification and immunoprecipitation. In-gel digestion with trypsin followed by peptide fingerprint analysis using matrix-assisted laser desorption ionization mass spectrometry and sequencing the major tryptic peptide obtained from liquid chromatography/mass spectrometry/mass spectrometry analysis identified E-FABP in the active fraction. Semi-quantitative Western blot analysis indicated that E-FABP in the cytosolic fraction of RBL-1 cells was present at approximately 1-3 pmol/10(6) cells. E-FABP (9 microm) was tested for its ability to stabilize LTA(4), and at 37 degrees C E-FABP was able to increase the half-life of LTA(4) from the previously reported half-life less than 3 s to a half-life of approximately 7 min. These results present a novel function for the well studied fatty acid-binding protein as a participant in leukotriene biosynthesis that permits LTA(4) to be available for further enzymatic processing in various cellular regions.
Highlights
Leukotrienes are a family of biologically active metabolites of arachidonic acid known to play important roles in multiple physiological and pathophysiological processes by acting as lipid mediators through specific G protein-coupled receptors [1, 2]
epithelial fatty acid-binding protein (E-fatty acid-binding proteins (FABPs)) (9 M) was tested for its ability to stabilize leukotriene A4 (LTA4), and at 37 °C E-FABP was able to increase the half-life of LTA4 from the previously reported half-life less than 3 s to a halflife of ϳ7 min. These results present a novel function for the well studied fatty acid-binding protein as a participant in leukotriene biosynthesis that permits LTA4 to be available for further enzymatic processing in various cellular regions
Studies of the fate of LTA4 produced within the human neutrophil revealed that greater than 50% of this lipid generated after cell activation is released to participate in the process of transcellular metabolism [13]
Summary
Leukotrienes are a family of biologically active metabolites of arachidonic acid known to play important roles in multiple physiological and pathophysiological processes by acting as lipid mediators through specific G protein-coupled receptors [1, 2] The biosynthesis of these 20 carbon fatty acids is regulated within cells that express 5-lipoxygenase, the first committed enzyme of the leukotriene cascade. Once LTA4 is formed and released from the active site of 5-lipoxygenase, a competitive nonenzymatic reaction with water can lead to the hydrolysis of the epoxide in a reaction in buffer measured to have a half-life of less than 30 s at 37 °C [7] through formation of a carbocation intermediate [8] The products of this nonenzymatic hydrolysis reaction include biologically inactive but chemical stable isomers of LTB4 including the ⌬6-trans-5,12-dihydroxyeicosatetraenoic acid and 5,6-dihydroxyeicosatetraenoic acid [8]. Our findings identify epithelial fatty acid-binding protein as an important molecule for stabilizing LTA4 in these cells
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