Characterization of Human Liver Leukotriene B(4) Omega-Hydroxylase P450 (CYP4F2)

J Biochem. 2000 Jun;127(6):1047-52. doi: 10.1093/oxfordjournals.jbchem.a022696.

Abstract

We previously reported the cloning of a human liver leukotriene B(4) (LTB(4)) omega-hydroxylase P450 designated CYP 4F2 [Kikuta et al. (1994) FEBS Lett. 348, 70-74]. However, the properties of CYP 4F2 remain poorly defined. The preparation solubilized using n-octyl-beta-D-glucopyranoside from microsomes of CYP 4F2-expressing yeast cells catalyzes v- hydroxylation of LTB(4), 6-trans-LTB(4), lipoxin A(4), 8-hydroxyeicosatetraenoate, 12-hydroxyeicosatetraenoate, and 12-hydroxystearate in the presence of rabbit liver NADPH-P450 reductase. In addition, the enzyme shows ethoxycoumarin O-deethylase and p-nitroanisole O-demethylase activities. The enzyme was purified to apparent electrophoretic homogeneity from yeast cells by sequential chromatography of solubilized microsomes through amino-n-hexyl-Sepharose 4B, DEAE-HPLC, and hydroxylapatite HPLC columns. The final preparation showed a specific content of 11.1 nmol of P450/mg of protein, with an apparent molecular mass of 56.3 kDa. CYP 4F2 was distinguished from the closely homologous CYP 4F3 (human neutrophil LTB(4) omega-hydroxylase) by its much higher K(m) for LTB(4), inability to omega-hydroxylate lipoxin B(4), and extreme instability.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Chromatography, High Pressure Liquid
  • Cytochrome P-450 Enzyme System / chemistry*
  • Cytochrome P-450 Enzyme System / genetics
  • Cytochrome P450 Family 4
  • Electrophoresis, Polyacrylamide Gel
  • Enzyme Stability
  • Humans
  • Hydroxylation
  • Kinetics
  • Leukotriene B4 / metabolism*
  • Liver / enzymology*
  • Microsomes / enzymology
  • Molecular Sequence Data
  • NADH, NADPH Oxidoreductases / metabolism
  • NADPH-Ferrihemoprotein Reductase
  • Neutrophils / enzymology
  • Rabbits
  • Recombinant Proteins / chemistry
  • Saccharomyces cerevisiae / enzymology
  • Saccharomyces cerevisiae / genetics
  • Sequence Homology, Amino Acid

Substances

  • Recombinant Proteins
  • Leukotriene B4
  • Cytochrome P-450 Enzyme System
  • Cytochrome P450 Family 4
  • CYP4F2 protein, human
  • NADH, NADPH Oxidoreductases
  • NADPH-Ferrihemoprotein Reductase