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

Biochemistry. 1997 Jun 3;36(22):6692-9. doi: 10.1021/bi963051a.

Abstract

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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Ammonium Sulfate
  • Animals
  • Arachidonate 12-Lipoxygenase / chemistry*
  • Arachidonate 12-Lipoxygenase / genetics*
  • Catalase / pharmacology
  • Chemical Precipitation
  • Chromatography, Ion Exchange
  • DNA, Complementary / genetics
  • Dithiothreitol / pharmacology
  • Escherichia coli / genetics
  • Gene Expression* / drug effects
  • Humans
  • Leukocytes / enzymology*
  • Lipoxygenase Inhibitors
  • Methionine*
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Recombinant Proteins / metabolism
  • Sequence Alignment
  • Structure-Activity Relationship
  • Swine

Substances

  • DNA, Complementary
  • Lipoxygenase Inhibitors
  • Recombinant Proteins
  • Methionine
  • Catalase
  • Arachidonate 12-Lipoxygenase
  • Ammonium Sulfate
  • Dithiothreitol