Leukocyte 12-lipoxygenase: expression, purification, and investigation of the role of methionine residues in turnover-dependent inactivation and 5,8,11,14-eicosatetraynoic acid inhibition.
Porcine leukocyte 12-lipoxygenase cDNA was cloned into the expression vectors pSE280, pSE380, and pSE420. pSE380 yielded the highest level of 12-lipoxygenase activity when these vectors were tested for expression in Escherichia coli Top10 cells. Optimal expression of the protein from this vector occurred in cells cultured at 30 degrees C and harvested 18 h following induction of expression by 0.5 mM isopropyl thiogalactoside (IPTG). The enzyme was purified from the 100000 g supernatant to 98% homogeneity by a combination of ammonium sulfate precipitation, anion exchange chromatography, and chromatofocusing. Addition of dithiothreitol and catalase to buffers at various steps in the purification protocol enabled the isolation of enzyme having a specific activity of 12 micromol min(-1) mg(-1). The recovery of purified protein from this expression system was 56%, resulting in a 109-fold purification. On the basis of amino acid sequence comparisons between mammalian 15- and 12-lipoxygenases, three methionine residues in the porcine leukocyte 12-lipoxygenase (M338L, M367V, and M562L) were targeted for mutation to assess their potential role in turnover-dependent inactivation and inhibition by 5,8,11,14-eicosatetraynoic acid (ETYA). The mutants were expressed and purified by the same procedure used for the wild-type enzyme. These amino acid changes did not significantly alter enzyme catalysis as judged by the kinetic constants Km and k(cat)/Km, nor did they affect the rate of turnover-dependent inactivation or inhibition by ETYA. The results indicate that these methionine residues do not play a pivotal role in catalysis, autoinactivation, or sensitivity to inhibition by acetylenic compounds.
Richards
K M
KM
Center in Molecular Toxicology, Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
Marnett
L J
LJ
D000595:Amino Acid Sequence; D000645:Ammonium Sulfate; D000818:Animals; D001092:Arachidonate 12-Lipoxygenase / Q000737:chemistry* / Q000235:genetics*; D002374:Catalase / Q000494:pharmacology; D011232:Chemical Precipitation; D002852:Chromatography, Ion Exchange; D018076:DNA, Complementary / Q000235:genetics; D004229:Dithiothreitol / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D007962:Leukocytes / Q000201:enzymology*; D016859:Lipoxygenase Inhibitors; D008715:Methionine; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D013552:Swine
D018076:DNA, Complementary; D016859:Lipoxygenase Inhibitors; D011994:Recombinant Proteins; D008715:Methionine; D002374:Catalase; D001092:Arachidonate 12-Lipoxygenase; D000645:Ammonium Sulfate; D004229:Dithiothreitol