The C2-like beta-barrel domain mediates the Ca2+-dependent resistance of 5-lipoxygenase activity against inhibition by glutathione peroxidase-1.
Recently, we reported that in crude enzyme preparations, a monocyte-derived soluble protein (M-DSP) renders 5-lipoxygenase (5-LO) activity Ca2+-dependent. Here we provide evidence that this M-DSP is glutathione peroxidase (GPx)-1. Thus, the inhibitory effect of the M-DSP on 5-LO could be overcome by the GPx-1 inhibitor mercaptosuccinate and by the broad spectrum GPx inhibitor iodoacetate, as well as by addition of 13(S)-hydroperoxy-9Z,11E-octadecadienoic acid (13(S)-HPODE). Also, the chromatographic characteristics and the estimated molecular mass (80-100 kDa) of the M-DSP fit to GPx-1 (87 kDa), and GPx-1, isolated from bovine erythrocytes, mimicked the effects of the M-DSP. Intriguingly, only a trace amount of thiol (10 micro M GSH) was required for reduction of 5-LO activity by GPx-1 or the M-DSP. Moreover, the requirement of Ca2+ allowing 5-LO product synthesis in various leukocytes correlated with the respective GPx-1 activities. Mutation of the Ca2+ binding sites within the C2-like domain of 5-LO resulted in strong reduction of 5-LO activity by M-DSP and GPx-1, also in the presence of Ca2+. In summary, our data suggest that interaction of Ca2+ at the C2-like domain of 5-LO protects the enzyme against the effect of GPx-1. Apparently, in the presence of Ca2+, a low lipid hydroperoxide level is sufficient for 5-LO activation.
Bürkert
Eva
E
Institute of Pharmaceutical Chemistry, University of Frankfurt, Marie-Curie-Strasse 9, D-60439 Frankfurt, Germany.
Arnold
Christina
C
Hammarberg
Tove
T
Rådmark
Olof
O
Steinhilber
Dieter
D
Werz
Oliver
O
D000818:Animals; D001094:Arachidonate 5-Lipoxygenase / Q000737:chemistry* / Q000378:metabolism; D001393:Azoles / Q000494:pharmacology; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D002417:Cattle; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005979:Glutathione Peroxidase / Q000378:metabolism*; D006801:Humans; D054833:Isoindoles; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D008041:Linoleic Acids / Q000494:pharmacology; D008054:Lipid Peroxides / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D016566:Organoselenium Compounds / Q000494:pharmacology; D010545:Peroxides / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D013870:Thiomalates / Q000494:pharmacology
D001393:Azoles; D004791:Enzyme Inhibitors; D054833:Isoindoles; D008041:Linoleic Acids; D008054:Lipid Peroxides; D016566:Organoselenium Compounds; D010545:Peroxides; D011994:Recombinant Proteins; D013870:Thiomalates; C038649:13-hydroperoxy-9,11-octadecadienoic acid; C042986:ebselen; C046062:2-thiomalic acid; D005979:Glutathione Peroxidase; D001094:Arachidonate 5-Lipoxygenase; D002118:Calcium