Purification and characterization of recombinant human neutrophil leukotriene B4 omega-hydroxylase (cytochrome P450 4F3).
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Kikuta Y, Kusunose E, Sumimoto H, Mizukami Y, Takeshige K, Sakaki T, Yabusaki Y, Kusunose M
Purification and characterization of recombinant human neutrophil leukotriene B4 omega-hydroxylase (cytochrome P450 4F3).
Arch Biochem Biophys. 1998 Jul 15;355(2):201-5. doi: 10.1006/abbi.1998.0724.
- PubMed ID
- 9675028 [ View in PubMed]
- Abstract
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.