Stabilization of leukotriene A4 by epithelial fatty acid-binding protein in the rat basophilic leukemia cell.
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.
Dickinson Zimmer
Jennifer S
JS
Department of Pharmacology, University of Colorado Health Sciences Center, Denver, Colorado 80262.
Voelker
Dennis R
DR
Bernlohr
David A
DA
Murphy
Robert C
RC
D000818:Animals; D015153:Blotting, Western; D002352:Carrier Proteins / Q000032:analysis / Q000494:pharmacology / Q000502:physiology*; D002851:Chromatography, High Pressure Liquid; D003600:Cytosol / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D005136:Eye Proteins; D050556:Fatty Acid-Binding Proteins; D006207:Half-Life; D007163:Immunosorbent Techniques; D015471:Leukemia, Basophilic, Acute / Q000378:metabolism*; D017572:Leukotriene A4 / Q000737:chemistry / Q000378:metabolism*; D013058:Mass Spectrometry; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010449:Peptide Mapping; D051381:Rats; D020539:Sequence Analysis, Protein; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000378:metabolism; D014407:Tumor Cells, Cultured
D002352:Carrier Proteins; D005136:Eye Proteins; C107356:Fabp5 protein, rat; D050556:Fatty Acid-Binding Proteins; D017572:Leukotriene A4; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D014357:Trypsin