Reversible membrane association of neutrophil 5-lipoxygenase is accompanied by retention of activity and a change in substrate specificity.
Ionophore activation of the human polymorphonuclear neutrophil results in eicosanoid synthesis and the accumulation of inactive 5-lipoxygenase in a membrane compartment. We report here that inhibition of self-inactivation of 5-lipoxygenase in ionophore-treated neutrophils with the reversible inhibitor zileuton, results in the accumulation of active 5-lipoxygenase in the membrane fraction. In zileuton plus ionophore-treated cells, 77% of the specific activity of the cytosolic enzyme from resting cells was diverted to the membrane fraction compared to 22% of the activity translocated when ionophore alone was used to activate the neutrophils. Accumulation of active membrane-associated 5-lipoxygenase was inhibited and reversed by the 5-lipoxygenase translocation inhibitor MK-886. The membrane-associated 5-lipoxygenase was two times more efficient in the production of leukotriene A4 from arachidonate-derived 5-hydroperoxyeicosatetraenoic acid than the cytosolic enzyme. Unlike the cytosolic enzyme, membrane-associated 5-lipoxygenase could metabolize 12(S)- and 15(S)-hydroxyeicosatetraenoic acid to 5(S),12(S)- and 5(S),15(S)-dihydroxyeicosatetraenoic acid, respectively. The ability to metabolize hydroxy fatty acids was dependent upon 5-lipoxygenase-activating protein association, but was lost if 5-lipoxygenase was eluted from the membrane by MK-886. These studies reveal for the first time that significant quantities of active 5-lipoxygenase can be detected in the membrane fraction of activated neutrophils and show that membrane association can alter the substrate specificity of 5-lipoxygenase which is further evidence for the role of the membrane-associated enzyme in the synthesis of 5-lipoxygenase metabolites.
Hill
E
E
National Jewish Center for Immunology and Respiratory Medicine, Denver, Colorado 80206.
Maclouf
J
J
Murphy
R C
RC
Henson
P M
PM
D001094:Arachidonate 5-Lipoxygenase / Q000378:metabolism*; D016718:Arachidonic Acid / Q000378:metabolism; D000001:Calcimycin / Q000494:pharmacology; D002451:Cell Compartmentation; D002462:Cell Membrane / Q000201:enzymology; D003600:Cytosol / Q000201:enzymology; D006801:Humans; D006893:Hydroxyeicosatetraenoic Acids / Q000378:metabolism; D006918:Hydroxyurea / Q000031:analogs & derivatives / Q000494:pharmacology; D066298:In Vitro Techniques; D007211:Indoles / Q000494:pharmacology; D015289:Leukotrienes / Q000378:metabolism; D009504:Neutrophils / Q000201:enzymology*; D013379:Substrate Specificity; D002722:Chlorobenzenes
D001094:Arachidonate 5-Lipoxygenase; D016718:Arachidonic Acid; D000001:Calcimycin; D006893:Hydroxyeicosatetraenoic Acids; D006918:Hydroxyurea; D007211:Indoles; D015289:Leukotrienes; C060893:MK-886; D002722:Chlorobenzenes; C025348:arachidonic acid 5-hydroperoxide; C063449:zileuton