Purification and characterization of recombinant human neutrophil leukotriene B4 omega-hydroxylase (cytochrome P450 4F3).
Recombinant human neutrophil leukotriene B4 (LTB4) omega-hydroxylase (cytochrome P450 4F3) has been purified to a specific content of 14. 8 nmol of P450/mg of protein from yeast cells. The purified enzyme was homogenous as judged from the SDS-PAGE, with an apparent molecular weight of 55 kDa. The enzyme catalyzed the omega-hydroxylation of LTB4 with a Km of 0.64 microM and Vmax of 34 nmol/min/nmol of P450 in the presence of rabbit hepatic NADPH-P450 reductase and cytochrome b5. Furthermore, various eicosanoids such as 20-hydroxy-LTB4, 6-trans-LTB4, lipoxin A4, lipoxin B4, 5-HETE and 12-HETE, and 12-hydroxy-stearate and 12-hydroxy-oleate were efficiently omega-hydroxylated, although their Km values were much higher than that of LTB4. In contrast, no activity was detected toward laurate, palmitate, arachidonate, 15-HETE, prostaglandin A1, and prostaglandin E1, all of which are excellent substrates for the CYP4A fatty acid omega-hydroxylases. This is the first time human neutrophil LTB4 omega-hydroxylase has been isolated in a highly purified state and characterized especially with respect to its substrate specificity.
Department of Food Science and Technology, Fukuyama University, Hiroshima, Fukuyama, 7290292, Japan.
Y
Y
Kikuta
E
E
Kusunose
H
H
Sumimoto
Y
Y
Mizukami
K
K
Takeshige
T
T
Sakaki
Y
Y
Yabusaki
M
M
Kusunose
D001483:Base Sequence; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification*; D000072470:Cytochrome P450 Family 4; D004789:Enzyme Activation; D005822:Genetic Vectors / Q000378:metabolism; D006801:Humans; D007962:Leukocytes; D007975:Leukotriene B4 / Q000031:analogs & derivatives / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D013053:Spectrophotometry; D013379:Substrate Specificity
D011994:Recombinant Proteins; D007975:Leukotriene B4; C031571:5,12,20-trihydroxy-6,8,10,14-eicosatetraenoic acid; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D000072470:Cytochrome P450 Family 4; C040780:leukotriene B4 20-hydroxylase