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 differen tiation of hematopoietic cells are commonly regarded as cytosolic enzy mes, Studying the interaction of the purified rabbit reticulocyte 15-l ipoxygenase with various types of biomembranes, we found that the enzy me binds to biomembranes when calcium is present in the incubation mix ture, Under these conditions, an oxidation of the membrane lipids was observed. The membrane binding was reversible and led to an increase i n the fatty acid oxygenase activity of the enzyme. To find out whether such a membrane binding also occurs in vivo, we investigated the intr acellular localization of the enzyme in stimulated and resting hematop oietic 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. T his membrane binding was also reversible and the detection of specific lipoxygenase products in the membrane lipids indicated the in vivo ac tivity of the enzyme on endogenous substrates, Immunoelectron microsco py showed that in interleukin-4 -treated monocytes, the 15-lipoxygenas e was localized in the cytosol, but also at the inner side of the plas ma membrane and at the cytosolic side of intracellular vesicles. Here again, cell fractionation studies confirmed the in vivo membrane bindi ng of the enzyme. In human eosinophils, which constitutively express t he 15-lipoxygenase, the membrane bound share of the enzyme was augment ed when the cells were stimulated with calcium ionophore. Only under t hese conditions, specific lipoxygenase products were detected in the m embrane lipids, These data suggest that in hematopoietic cells the cyt osolic 15-lipoxygenase translocates reversibly to the cellular membran es, This translocation, which increases the fatty acid oxygenase activ ity of the enzyme, is calcium-dependent, but may not require a special docking protein. (C) 1998 by The American Society of Hematology.