5-LIPOXYGENASE AND 5-LIPOXYGENASE ACTIVATING PROTEIN ARE LOCALIZED INTHE NUCLEAR-ENVELOPE OF ACTIVATED HUMAN-LEUKOCYTES

The intracellular distribution of the enzyme 5-lipoxygenase (5-LO) and 5-lipoxygenase-activating protein (FLAP) in resting and ionophore-act ivated human leukocytes has been determined using immuno-electronmicro scopic labeling of ultrathin frozen sections and subcellular fractiona tion techniques. 5-LO is a 78-kD protein that catalyzes the conversion of arachidonic acid to leukotrienes. FLAP is an 18-kD membrane bound protein that is essential for leukotriene synthesis in cells. In respo nse to ionophore stimulation, 5-LO translocates from a soluble to a se dimentable fraction of cell homogenates. In activated leukocytes, both FLAP and 5-LO were localized in the lumen of the nuclear envelope. Ne ither protein could be detected in any other cell compartment or along the plasma membrane. In resting cells, the FLAP distribution was iden tical to that observed in activated cells. In addition, subcellular fr actionation techniques showed >83% of immunoblot-detectable FLAP prote in and approximately 64% of the FLAP ligand binding activity was found in the nuclear membrane fraction. A fractionation control demonstrate d that a plasma membrane marker, detected by a monoclonal antibody PMN 13F6, was not detectable in the nuclear membrane fraction. In contrast to FLAP, 5-LO in resting cells could not be visualized along the nucl ear envelope. Except for weak labeling of the euchromatin region of th e nucleus, 5-LO could not be readily detected in any other cellular co mpartment. These results demonstrate that the nuclear envelope is the intracellular site at which 5-LO and FLAP act to metabolize arachidoni c acid, and that ionophore activation of neutrophils and monocytes res ults in the translocation of 5-LO from a nonsedimentable location to t he nuclear envelope.