Membrane Translocation of 15-Lipoxygenase in Hematopoietic Cells Is Calcium-Dependent and Activates the Oxygenase Activity of the Enzyme

Abstract

Mammalian 15-lipoxygenases, which have been implicated in the differentiation of hematopoietic cells are commonly regarded as cytosolic enzymes. Studying the interaction of the purified rabbit reticulocyte 15-lipoxygenase with various types of biomembranes, we found that the enzyme binds to biomembranes when calcium is present in the incubation mixture. Under these conditions, an oxidation of the membrane lipids was observed. The membrane binding was reversible and led to an increase in the fatty acid oxygenase activity of the enzyme. To find out whether such a membrane binding also occurs in vivo, we investigated the intracellular localization of the enzyme in stimulated and resting hematopoietic cells by immunoelectron microscopy, cell fractionation studies and activity assays. In rabbit reticulocytes, the 15-lipoxygenase was localized in the cytosol, but also bound to intracellular membranes. This membrane binding was also reversible and the detection of specific lipoxygenase products in the membrane lipids indicated the in vivo activity of the enzyme on endogenous substrates. Immunoelectron microscopy showed that in interleukin-4 –treated monocytes, the 15-lipoxygenase was localized in the cytosol, but also at the inner side of the plasma membrane and at the cytosolic side of intracellular vesicles. Here again, cell fractionation studies confirmed the in vivo membrane binding of the enzyme. In human eosinophils, which constitutively express the 15-lipoxygenase, the membrane bound share of the enzyme was augmented when the cells were stimulated with calcium ionophore. Only under these conditions, specific lipoxygenase products were detected in the membrane lipids. These data suggest that in hematopoietic cells the cytosolic 15-lipoxygenase translocates reversibly to the cellular membranes. This translocation, which increases the fatty acid oxygenase activity of the enzyme, is calcium-dependent, but may not require a special docking protein.

15‐Lipoxygenase‐1 activates tumor suppressor p53 independent of enzymatic activity

“…Although 15‐LOX‐1 is a cytosolic enzyme, it can associate with the cell membrane 35. For example, the rabbit reticulocyte‐type 15‐LOX (15‐LOX‐1) associates with biomembranes in a calcium‐dependent manner 36. 15‐LOX‐1 also has been found both in the cytosol and the plasma membrane in nonstimulated human dendritic cells in the absence of calcium 37.…”

12‐Lipoxygenase plays a key role in cell death caused by glutathione depletion and arachidonic acid in rat oligodendrocytes

“…5A). Activation of LOXs is usually accompanied by translocation to membranes (Baba et al ., 1989; Hagmann et al ., 1993; Brock et al ., 1995; Brinckmann et al ., 1998). We observed translocation of 12‐LOX out of the nucleus in preOLs following cystine deprivation (Fig.…”

“…Activation of 5‐ and 12‐LOX has been shown to be accompanied by translocation to various cellular membranes, depending on the cell types (Baba et al ., 1989; Hagmann et al ., 1993; Brock et al ., 1995; Brinckmann et al ., 1998). To further characterize the effect of cystine deprivation on 12‐LOX activation, we investigated the distribution of 12‐LOX in preOLs using immunocytochemistry.…”

A re‐evaluation of 9‐HODE activity at TRPV1 channels in comparison with anandamide: enantioselectivity and effects at other TRP channels and in sensory neurons

“…These studies led to the recent discovery of a new family of endogenous TRPV1 ligands, 9‐hydroxyoctadecadienoic (9‐HODE) and 13‐hydroxyoctadecadienoic (13‐HODE) acid. Indeed, calcium influx caused by peripheral inflammation may activate lipoxygenases that can oxidize the ω‐6 fatty acid, linoleic acid (Brinckmann et al ., ), and lead to the production of 9‐ and 13‐HODE, which evoke mechanical allodynia following spinal administration (Patwardhan et al ., ). These putative endogenous TRPV1 agonists are also formed on exposure of cell plasma membranes to noxious heat, thus contributing to the thermal responsiveness of this channel (Patwardhan et al ., ).…”