Characterization of human liver leukotriene B(4) omega-hydroxylase P450 (CYP4F2).
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.
Kikuta
Y
Y
Department of Applied Biological Science, Faculty of Engineering, Fukuyama University, Fukuyama, Hiroshima 729-0292, Japan. kikuta@fubac.fukuyama-u.ac.jp
Kusunose
E
E
Kusunose
M
M
D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics; D000072470:Cytochrome P450 Family 4; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006801:Humans; D006900:Hydroxylation; D007700:Kinetics; D007975:Leukotriene B4 / Q000378:metabolism*; D008099:Liver / Q000201:enzymology*; D008861:Microsomes / Q000201:enzymology; D008969:Molecular Sequence Data; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase; D009504:Neutrophils / Q000201:enzymology; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D017386:Sequence Homology, Amino Acid
D011994:Recombinant Proteins; D007975:Leukotriene B4; D003577:Cytochrome P-450 Enzyme System; D000072470:Cytochrome P450 Family 4; C117480:CYP4F2 protein, human; D009247:NADH, NADPH Oxidoreductases; D009251:NADPH-Ferrihemoprotein Reductase