title: Activity and protein distribution of 12-lipoxygenase in HEL cells: induction of membrane-association by phorbol ester TPA, modulation of activity by glutathione and 13-HPODE, and Ca(2+)-dependent translocation to membranes.
abstract: The understanding of the intracellular regulation of 12-lipoxygenase requires a knowledge of the distribution of both enzyme protein and its activity. In human erythroleukemia cells, the membrane fraction contains about 90% of the total cellular 12-lipoxygenase activity, whereas only approximately 10% of 12-lipoxygenase activity resides in the cytosol. However, the majority of the cellular 12-lipoxygenase protein is found in the cytosol. Pretreatment of cells for 0-3 days with 160 nM TPA caused a marked, time-dependent increase in membrane-bound 12-lipoxygenase activity and protein, respectively. In contrast, the cytosolic amount of 12-lipoxygenase protein and activity, respectively, were minimally altered by this TPA treatment. Recombining the active membrane fraction with cytosol resulted in no significant inhibition of its 12-lipoxygenase activity, but the addition of GSH to the membrane fraction inhibited 12-lipoxygenase activity in a dose-dependent manner. On the other hand, the cytosolic enzyme can be rendered active in the presence of 1 microM 13-hydroperoxyoctadecadienoic acid. In HEL cell homogenates, a partial translocation of the cytosolic enzyme to the membrane takes place in a Ca(2+)-dependent manner, resulting in an increase in membrane-associated 12-lipoxygenase activity and a concomitant decrease in cytosolic 12-lipoxygenase activity above 0.1 microM Ca2+.
authors.affiliation: Dept. of Radiation Oncology, Wayne State University, Detroit, MI 48202.
authors.forename: W
authors.initials: W
authors.lastname: Hagmann
authors.forename: D
authors.initials: D
authors.lastname: Kagawa
authors.forename: C
authors.initials: C
authors.lastname: Renaud
authors.forename: K V
authors.initials: KV
authors.lastname: Honn
mesh_terms: D001092:Arachidonate 12-Lipoxygenase / Q000378:metabolism*; D001692:Biological Transport / Q000187:drug effects; D002118:Calcium / Q000494:pharmacology*; D002462:Cell Membrane / Q000201:enzymology*; D003600:Cytosol / Q000201:enzymology; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D005978:Glutathione / Q000494:pharmacology*; D006801:Humans; D015151:Immunoblotting; D004915:Leukemia, Erythroblastic, Acute / Q000201:enzymology; D008041:Linoleic Acids / Q000494:pharmacology*; D008054:Lipid Peroxides; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
chemical_list: D008041:Linoleic Acids; D008054:Lipid Peroxides; C038649:13-hydroperoxy-9,11-octadecadienoic acid; D001092:Arachidonate 12-Lipoxygenase; D005978:Glutathione; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium; D004121:Dimethyl Sulfoxide