Membrane translocation of 15-lipoxygenase in hematopoietic cells is calcium-dependent and activates the oxygenase activity of the enzyme.
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.
Brinckmann
R
R
Institute of Biochemistry Pathology and Dermatological Clinic, University Clinics Charité, Humboldt University, Berlin, Germany.
Schnurr
K
K
Heydeck
D
D
Rosenbach
T
T
Kolde
G
G
Kühn
H
H
D000818:Animals; D001093:Arachidonate 15-Lipoxygenase / Q000378:metabolism*; D002118:Calcium / Q000502:physiology*; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D003600:Cytosol / Q000201:enzymology; D004789:Enzyme Activation; D004804:Eosinophils / Q000201:enzymology* / Q000648:ultrastructure; D005230:Fatty Acids, Nonesterified / Q000378:metabolism*; D006801:Humans; D007124:Immunoenzyme Techniques; D007150:Immunohistochemistry; D007963:Leukocytes, Mononuclear / Q000201:enzymology* / Q000648:ultrastructure; D016253:Microscopy, Immunoelectron; D010084:Oxidation-Reduction; D011817:Rabbits; D012156:Reticulocytes / Q000201:enzymology* / Q000648:ultrastructure; D013347:Subcellular Fractions
D005230:Fatty Acids, Nonesterified; D001093:Arachidonate 15-Lipoxygenase; D002118:Calcium