LOCALIZATION OF 5-LIPOXYGENASE TO THE NUCLEUS OF UNSTIMULATED RAT BASOPHILIC LEUKEMIA-CELLS

Arachidonate metabolism by 5-lipoxygenase (5-LO) coincides with the tr anslocation of the enzyme from a soluble to a pelletable fraction in t horoughly disrupted granulocytic cells. While immunoelectron microscop y has identified the nuclear membrane as the site at which 5-LO, as we ll as 5-LO activating protein (FLAP), are localized in activated cells , the locale of soluble 5-LO in unstimulated cells could not be establ ished by this technique. We asked whether the nucleus might also be th e site for soluble 5-LO in unstimulated cells, and utilized rat basoph ilic leukemia (RBL) cells as model granulocytic cells to address this question. Using three different techniques to disrupt cells while leav ing nuclei intact (mild nitrogen cavitation, Dounce homogenization, an d detergent lysis), immunoblot analysis indicated abundant 5-LO in iso lated nuclei. Within purified nuclei, 5-LO existed in two pools: a sol uble pool that was readily released upon nuclear disruption and a boun d pool that was not removed by 300 mM NaCl treatment. In all cases, 5- LO was also found in cytosolic and non-nuclear membrane fractions. Ind irect immunofluorescent microscopy confirmed the presence of abundant 5-LO within the nucleus with minimal extranuclear signal in most cells . However, a minority of cells, characterized by condensed chromatin, showed no nuclear-associated staining with increased cytoplasmic stain ing for 5-LO. This suggested that some of the cytosolic 5-LO found by cell fractionation resulted from these dividing cells. When the contri bution from dividing cells was minimized, either by overnight serum de privation or by isolating cytoplasts of nucleus-containing cells, 5-LO was prominent in the nuclear fraction but negligible in the cytosolic fraction. In contrast to this distribution in RBL cells, 5-LO in unst imulated human neutrophils was predominantly cytosolic, by both immuno blot and immunofluorescence analyses. In both RBL cells and human neut rophils, FLAP was localized at the nuclear membrane and the endoplasmi c reticulum. These data provide the first evidence for the localizatio n of 5-LO in unstimulated granulocytic cells. The finding that a subst antial proportion of enzyme is localized within the nucleus of unstimu lated RBL cells suggests potentially novel roles for 5-LO or its produ cts within the nucleus.