Oxidation of
low density lipoprotein (
LDL) is the key step for the development of
atherosclerosis. The 12/
15-lipoxygenase expressed in macrophages is capable of oxygenating
linoleic acid esterified to
cholesterol in the
LDL particle, and thus this
enzyme is presumed to initiate
LDL oxidation. We recently reported that
LDL receptor-related protein (LRP) was required for the
enzyme-mediated
LDL oxidation by macrophages and suggested the selective uptake of
cholesterol ester from
LDL to the plasma membrane (Xu, W., Takahashi, Y., Sakashita, T., Iwasaki, T., Hattori, H., and Yoshimoto. T. (2001) J. Biol. Chem. 276, 36454-36459). To elucidate precise mechanisms of
lipoxygenase-mediated
LDL oxidation, we investigated the intracellular localization of 12/
15-lipoxygenase. The 12/
15-lipoxygenase was predominantly detected in cytosol of resting peritoneal macrophages and of macrophage-like J774A.1 cells permanently transfected with the
cDNA for the
enzyme. When the cells were treated with
LDL and subjected to subcellular fractionation, the 12/
15-lipoxygenase was detected in the membranes with a concomitant decrease in cytosol as shown by Western blot analysis. The levels of the
enzyme associated with the membrane reached maximum in 15 min after
LDL addition and then decreased. However, the enzymatic activity of 12/
15-lipoxygenase in the membrane fraction was very weak even after
LDL treatment. This fact supports the suicide inactivation of the
enzyme by the oxygenation of
cholesterol ester transferred from the
LDL particle to the plasma membrane. Immunohistochemical analysis using an antibody against 12/
15-lipoxygenase revealed that the plasma membrane was the major site of the
enzyme translocation by the
LDL treatment.
LDL-dependent 12/
15-lipoxygenase translocation was inhibited by a blocking antibody against LRP. Furthermore, an
enzyme translocation inhibitor, L655238, inhibited the
LDL oxidation caused by the 12/
15-lipoxygenase. We propose that
cholesterol ester selectively transferred from the
LDL particle to the plasma membrane via LRP is oxygenated by 12/
15-lipoxygenase translocated to this membrane.