5-hydroxy-6-8-11-14-eicosatetraenoic-acid and Leukemia--Promyelocytic--Acute

We report that leukotriene A4, the electrophilic product of 5-lipoxygenase catalysis, irreversibly inactivates the enzyme. Leukotriene A4 inhibits 5-hydroxyeicosatetraenoic acid formation by human neutrophils and differentiated granulocytic HL-60 cells in a concentration-dependent manner with IC50 values = 22.4 +/- 2.5 and 29.0 +/- 8.0 microM, respectively. Recovery of cellular enzymatic activity is negligible (< 6%) following inactivation. Leukotriene A4 inactivates cellular 5-lipoxygenase without inhibiting its translocation from the cytosol to the membrane, suggesting that it impairs catalysis without impairing formation of the complex between 5-lipoxygenase and its membrane-associated activating protein. Consistent with this, leukotriene A4 inactivates purified 5-lipoxygenase from human neutrophils, via saturable, pseudo first-order kinetics with a rate constant, ki = 0.14 min-1 and a dissociation constant, Ki = 2.1 +/- 0.7 microM. Purified 5-lipoxygenase incubated with [3H]arachidonic acid incorporated a radiolabeled species that was not removed by electrophoresis under reduced denaturing conditions. Preincubation with leukotriene A4 diminished the incorporation of radiolabeled material, consistent with irreversible modification of 5-lipoxygenase by its metastable product, leukotriene A4. This unusual product inactivation mechanism may contribute to the decline in 5-lipoxygenase activity observed during catalysis.

Topics: Arachidonate 5-Lipoxygenase; Cell Differentiation; Cell Line; Humans; Hydroxyeicosatetraenoic Acids; Hydroxyurea; Kinetics; Leukemia, Promyelocytic, Acute; Leukocytes; Leukotriene A4; Lipoxygenase Inhibitors; Neutrophils; Tumor Cells, Cultured