Abstract
Endothelial cells contain leukotriene (LT) A4 hydrolase (LTA-H) as detected by Northern and Western blotting, but several studies have been unable to detect the activity of this enzyme. Since LTA-H could play a key role in determining what biologically active lipids are generated by activated endothelium during the inflammatory process, we studied possible mechanisms by which this enzyme may be regulated. We find that LTA-H is phosphorylated under basal conditions in human endothelial cells and in this state does not exhibit epoxide hydrolase activity (i.e. conversion of LTA4 to LTB4). LTA-H purified from endothelial cells is efficiently dephosphorylated by incubation with protein phosphatase-1 in the presence of an LTA-H peptide substrate and not at all in the absence of substrate. Under conditions that lead to dephosphorylation, protein phosphatase-1 activates the epoxide hydrolase activity of LTA-H. Using peptide mapping and site-directed mutagenesis, we have identified serine 415 as the site of phosphorylation of LTA-H by a kinase found in endothelial cell cytosol. In parallel, we have studied a human lung carcinoma cell line that expresses active LTA-H. Although these cells have cytosolic kinases that phosphorylate recombinant LTA-H, they do not target serine 415 and thus do not inhibit LTA-H activity. We believe that LTA-H is regulated in intact cells by a kinase/phosphatase cycle and further that the kinase in endothelial cells specifically recognizes and phosphorylates a regulatory site in the LTA-H.
Highlights
Leukotriene (LT)1 A4 hydrolase (LTA-H; endothelial cells (EC) 3.3.2.6) is a zinc metalloenzyme that catalyzes the hydrolysis of LTA4 to yield the dihydroxyarachidonic acid metabolite, LTB4 [1,2,3]
Presence of LTA-H in Endothelial Cells—Despite the fact that LTA-H activity was not detected in various endothelial cells, we tested for the presence of messenger RNA and enzyme protein in these cells
We retested these cells for the presence of LTA-H activity in an assay that measures the production of LTB4 from exogenous LTA4
Summary
Cell Culture—Human umbilical vein endothelial cells were cultured as previously reported [25]. Aminopeptidase Activity Assay—The aminopeptidase activity was determined by incubating recombinant LTA-H (1–2 g) with alaninep-nitroanilide (1 mM) in 50 mM Tris-Cl, pH 8.0, with bovine serum albumin (1 mg/ml) in 96-well microtiter plates at 37 °C for 30 min. [␥-32P]ATP (150 M, 20 –50 Ci; NEN Life Science Products) was added to the cell lysate (300 l; approximately 1.4 mg of total protein), and the reaction was begun by the addition of recombinant LTA-H. A 20-cycle PCR reaction was run containing deoxynucleotides (25 M each), pEX85 (100 ng), sense and antisense primers (50 pmol each), and Taq DNA polymerase (5 units) in PCR buffer (50 l of 10 mM Tris-Cl, pH 8.3, 50 mM KCl, 2.5 mM MgCl2, 0.001% gelatin). Postexperiment comparisons of individual means were performed using Tukey’s procedure
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