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J. Biol. Chem., Vol. 278, Issue 44, 42846-42853, October 31, 2003

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The C2-like -Barrel Domain Mediates the Ca²⁺-dependent Resistance of 5-Lipoxygenase Activity Against Inhibition by Glutathione Peroxidase-1^*

Eva Bürkert, Christina Arnold, Tove Hammarberg, Olof Rådmark, Dieter Steinhilber, and Oliver Werz¶

From the Institute of Pharmaceutical Chemistry, University of Frankfurt, Marie-Curie-Strasse 9, D-60439 Frankfurt, Germany and the Department of Medical Biochemistry and Biophysics, Division of Physiological, Chemistry II, Karolinska Institutet, S-171 77 Stockholm, Sweden

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 µM 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.

Received for publication, March 11, 2003 , and in revised form, July 30, 2003.

^* This study was supported by grants from the Fonds der Chemischen Industrie, the EU (LEUCHRON, QLRT-2000-01521), the Swedish Medical Research Council (03X-217), and the Deutsche Pharmazeutische Gesellschaft. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Institute of Pharmaceutical Chemistry, University of Frankfurt, Marie-Curie Str. 9, D-60439 Frankfurt, Germany. Tel.: 49-69-798-29337; Fax: 49-69-798-29323; E-mail: o.werz{at}pharmchem.uni-frankfurt.de.

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