title: 1-Oleoyl-2-acetylglycerol stimulates 5-lipoxygenase activity via a putative (phospho)lipid binding site within the N-terminal C2-like domain.
abstract: 5-Lipoxygenase (5-LO) catalysis is positively regulated by Ca2+ ions and phospholipids that both act via the N-terminal C2-like domain of 5-LO. Previously, we have shown that 1-oleoyl-2-acetylglycerol (OAG) functions as an agonist for human polymorphonuclear leukocytes (PMNL) in stimulating 5-LO product formation. Here we have demonstrated that OAG directly stimulates 5-LO catalysis in vitro. In the absence of Ca2+ (chelated using EDTA), OAG strongly and concentration-dependently stimulated crude 5-LO in 100,000 x g supernatants as well as purified 5-LO enzyme from PMNL. Also, the monoglyceride 1-O-oleyl-rac-glycerol and 1,2-dioctanoyl-sn-glycerol were effective, whereas various phospholipids did not stimulate 5-LO. However, in the presence of Ca2+, OAG caused no stimulation of 5-LO. Also, phospholipids or cellular membranes abolished the effects of OAG. As found previously for Ca2+, OAG renders 5-LO activity resistant against inhibition by glutathione peroxidase activity, and this effect of OAG is reversed by phospholipids. Intriguingly, a 5-LO mutant lacking tryptophan residues (Trp-13, -75, and -102) important for the binding of the 5-LO C2-like domain to phospholipids was not stimulated by OAG. We conclude that OAG directly stimulates 5-LO by acting at a phospholipid binding site located within the C2-like domain.
authors.affiliation: Institute of Pharmaceutical Chemistry, ZAFES, University of Frankfurt, D-60439 Frankfurt, Germany.
authors.forename: Christina
authors.initials: C
authors.lastname: Hörnig
authors.forename: Dana
authors.initials: D
authors.lastname: Albert
authors.forename: Lutz
authors.initials: L
authors.lastname: Fischer
authors.forename: Michael
authors.initials: M
authors.lastname: Hörnig
authors.forename: Olof
authors.initials: O
authors.lastname: Rådmark
authors.forename: Dieter
authors.initials: D
authors.lastname: Steinhilber
authors.forename: Oliver
authors.initials: O
authors.lastname: Werz
mesh_terms: D001094:Arachidonate 5-Lipoxygenase / Q000187:drug effects* / Q000378:metabolism; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology; D002478:Cells, Cultured; D004075:Diglycerides / Q000378:metabolism / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D005989:Glycerides / Q000494:pharmacology; D006801:Humans; D008563:Membrane Lipids / Q000494:pharmacology; D009504:Neutrophils / Q000201:enzymology; D010743:Phospholipids / Q000378:metabolism; D017434:Protein Structure, Tertiary
chemical_list: D004075:Diglycerides; D005989:Glycerides; D008563:Membrane Lipids; D010743:Phospholipids; C038237:1-oleoyl-2-acetylglycerol; D001094:Arachidonate 5-Lipoxygenase; D002118:Calcium