Purification and characterization of recombinant rat hepatic CYP4F1.
CYP4F1 was discovered by Chen and Hardwick (Arch. Biochem. Biophys. 300, 18-23, 1993) as a new CYP4 cytochrome P450 (P450) preferentially expressed in rat hepatomas. However, the catalytic function of this P450 remained poorly defined. We have purified recombinant CYP4F1 protein to a specific content of 12 nmol of P450/mg of protein from transfected yeast cells by chromatography of solubilized microsomes on an amino-n-hexyl Sepharose 4B column, followed by sequential HPLC on a DEAE column and two hydroxylapatite columns. The purified P450 was homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 53 kDa. The enzyme catalyzed the omega-hydroxylation of leukotriene B(4) with a K(m) of 134 microM and a V(max) of 6.5 nmol/min/nmol of P450 in the presence of rabbit hepatic NADPH-P450 reductase and cytochrome b(5). In addition, 6-trans-LTB(4), lipoxin A(4), prostaglandin A(1), and several hydroxyeicosatetraenoic acids (HETEs) were also omega-hydroxylated. Of several eicosanoids examined, 8-HETE was the most efficient substrate, with a K(m) of 18.6 microM and a V(max) of 15.8 nmol/min/nmol of P450. In contrast, no activity was detected toward lipoxin B(4), laurate, palmitate, arachidonate, and benzphetamine. The results suggest that CYP4F1 participates in the hepatic inactivation of several bioactive eicosanoids.
Kikuta
Y
Y
Department of Applied Biological Science, Fukuyama University, Hiroshima, Fukuyama, 7290292, Japan.
Kusunose
E
E
Ito
M
M
Kusunose
M
M
D000818:Animals; D006528:Carcinoma, Hepatocellular / Q000201:enzymology; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000072470:Cytochrome P450 Family 4; D006863:Hydrogen-Ion Concentration; D006893:Hydroxyeicosatetraenoic Acids / Q000378:metabolism; D006900:Hydroxylation; D007975:Leukotriene B4 / Q000378:metabolism; D044045:Lipoxins; D008099:Liver / Q000201:enzymology*; D008113:Liver Neoplasms / Q000201:enzymology; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000302:isolation & purification / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013379:Substrate Specificity
D006893:Hydroxyeicosatetraenoic Acids; D044045:Lipoxins; D009363:Neoplasm Proteins; D011994:Recombinant Proteins; C040527:lipoxin A4; D007975:Leukotriene B4; C047628:8-hydroxyeicosatetraenoic acid; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D000072470:Cytochrome P450 Family 4; C040780:leukotriene B4 20-hydroxylase