Publications

• Purification and characterization of recombinant human neutrophil leukotriene B4 omega-hydroxylase (cytochrome P450 4F3).

  Kikuta Y., Kusunose E., Sumimoto H., Mizukami Y., Takeshige K., Sakaki T., Yabusaki Y., Kusunose M.

  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 ... >> More

  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. << Less

  Arch. Biochem. Biophys. 355:201-205(1998) [PubMed] [EuropePMC]

  This publication is cited by 11 other entries.

• Expression and characterization of human cytochrome P450 4F11: Putative role in the metabolism of therapeutic drugs and eicosanoids.

  Kalsotra A., Turman C.M., Kikuta Y., Strobel H.W.

  We previously reported the cDNA cloning of a new CYP4F isoform, CYP4F11. In the present study, we have expressed CYP4F11 in Saccharomyces cerevisiae and examined its catalytic properties towards endogenous eicosanoids as well as some clinically relevant drugs. CYP4F3A, also known as a leukotriene ... >> More

  We previously reported the cDNA cloning of a new CYP4F isoform, CYP4F11. In the present study, we have expressed CYP4F11 in Saccharomyces cerevisiae and examined its catalytic properties towards endogenous eicosanoids as well as some clinically relevant drugs. CYP4F3A, also known as a leukotriene B4 omega-hydroxylase, was expressed in parallel for comparative purposes. Our results show that CYP4F11 has a very different substrate profile than CYP4F3A. CYP4F3A metabolized leukotriene B4, lipoxins A4 and B4, and hydroxyeicosatetraenoic acids (HETEs) much more efficiently than CYP4F11. On the other hand, CYP4F11 was a better catalyst than CYP4F3A for many drugs such as erythromycin, benzphetamine, ethylmorphine, chlorpromazine, and imipramine. Erythromycin was the most efficient substrate for CYP4F11, with a Km of 125 microM and Vmax of 830 pmol min(-1) nmol(-1) P450. Structural homology modeling of the two proteins revealed some interesting differences in the substrate access channel including substrate recognition site 2 (SRS2). The model of CYP4F11 presents a more open access channel that may explain the ability to metabolize large molecules like erythromycin. Also, some wide variations in residue size, charge, and hydrophobicity in the FG loop region may contribute to differences in substrate specificity and activity between CYP4F3A and CYP4F11. << Less

  Toxicol. Appl. Pharmacol. 199:295-304(2004) [PubMed] [EuropePMC]

  This publication is cited by 6 other entries.

• Catalytic activity and isoform-specific inhibition of rat cytochrome p450 4F enzymes.

  Xu F., Falck J.R., Ortiz de Montellano P.R., Kroetz D.L.

  Arachidonic acid is omega-hydroxylated to 20-hydroxyeicosatetraenoic acid (20-HETE), which has effects on vasoactivity and renal tubular transport and has been implicated in the regulation of blood pressure. Cytochrome p450 (p450) 4A isoforms are generally considered the major arachidonic acid ome ... >> More

  Arachidonic acid is omega-hydroxylated to 20-hydroxyeicosatetraenoic acid (20-HETE), which has effects on vasoactivity and renal tubular transport and has been implicated in the regulation of blood pressure. Cytochrome p450 (p450) 4A isoforms are generally considered the major arachidonic acid omega-hydroxylases; however, little is known about the role of rat CYP4F isoforms in 20-HETE formation. The rat CYP4F isoforms, CYP4F1, CYP4F4, CYP4F5, and CYP4F6, were heterologously expressed in Escherichia coli, and their substrate specificity in fatty acid metabolism was characterized. Substrate-binding assays indicated that leukotriene B(4) (LTB(4)) and arachidonic acid bound CYP4F1 and CYP4F4 in a type-I manner with a K(s) of 25 to 59 microM, and lauric acid bound CYP4F4 poorly. Reconstituted CYP4F1 and CYP4F4 catalyzed the omega-hydroxylation of LTB(4) with a K(m) of 24 and 31 microM, respectively, and CYP4F5 had minor activity in LTB(4) metabolism. Importantly, CYP4F1 and CYP4F4 catalyzed the omega-hydroxylation of arachidonic acid with an apparent k(cat) of 9 and 11 min(-1), respectively. Lauric acid was a poor substrate for all of the CYP4F isoforms, and CYP4F6 had no detectable fatty acid omega-hydroxylase activity. The p450 omega-hydroxylase inhibitors 17-octadecynoic acid, 10-undecynyl sulfate, and N-methylsulfonyl-12,12-dibromododec-11-enamide showed isoform-specific inhibition of CYP4F1- and CYP4F4-catalyzed omega-hydroxylation of arachidonic acid and potency differences between the CYP4A and CYP4F isoforms. These data support a significant role for CYP4F1 and CYP4F4 in the formation of 20-HETE and identify p450 inhibitors that can be used to understand the relative contribution of the CYP4A and CYP4F isoforms to renal 20-HETE formation. << Less

  J. Pharmacol. Exp. Ther. 308:887-895(2004) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Protein expression, characterization, and regulation of CYP4F4 and CYP4F5 cloned from rat brain.

  Kawashima H., Kusunose E., Thompson C.M., Strobel H.W.

  We previously reported the cDNA cloning of three new forms of P450, CYP4F4, CYP4F5, and CYP4F6, from a rat brain cDNA library. In the present study, we expressed CYP4F4 and CYP4F5 in Escherichia coli using the pCWOri expression vector with a modification of their N-terminal amino acid sequences an ... >> More

  We previously reported the cDNA cloning of three new forms of P450, CYP4F4, CYP4F5, and CYP4F6, from a rat brain cDNA library. In the present study, we expressed CYP4F4 and CYP4F5 in Escherichia coli using the pCWOri expression vector with a modification of their N-terminal amino acid sequences and the incorporation of a C-terminal [His]4 tag to aid in purification. CYP4F5 recombinant protein was purified to a specific content of 7.7 nmol/mg protein from the membrane fraction of E. coli and showed omega-hydroxylation activity toward leukotriene B4 (LTB4), a chemical mediator of inflammation. On the other hand, the solubilized membrane fraction of CYP4F4-expressed recombinant protein catalyzed the omega-hydroxylation of prostaglandin A1, prostaglandin E1, and 6-trans-LTB4 as well as LTB4. The effects of the peroxisome proliferator, clofibrate, on mRNA expression of CYP4F4, 4F5, and 4F6 were studied by Northern blot analysis. The expression levels of the mRNA of these CYP4Fs were shown to be reduced by clofibrate in liver. << Less

  Arch. Biochem. Biophys. 347:148-154(1997) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• Cloning and expression of a novel form of leukotriene B4 omega-hydroxylase from human liver.

  Kikuta Y., Kusunose E., Kondo T., Yamamoto S., Kinoshita H., Kusunose M.

  We have isolated and sequenced a cDNA for human liver LTB4 omega-hydroxylase. The cDNA encoded a protein of 520 amino acids with a molecular weight of 59,853 Da. The cDNA-deduced amino acid sequence showed 87.3% homology to that of human polymorphonuclear leukocytes (PMN) LTB4 omega-hydroxylase (C ... >> More

  We have isolated and sequenced a cDNA for human liver LTB4 omega-hydroxylase. The cDNA encoded a protein of 520 amino acids with a molecular weight of 59,853 Da. The cDNA-deduced amino acid sequence showed 87.3% homology to that of human polymorphonuclear leukocytes (PMN) LTB4 omega-hydroxylase (CYP4F3). Northern blot analysis revealed that the mRNA hybridized to the specific cDNA fragment is expressed in human liver, but not in human PMN. The microsomes from yeast cells transfected with the cDNA catalyzed the omega-hydroxylation of LTB4 with a Km of 44.8 microM. These results clearly show that a new form of the CYP4F LTB4 omega-hydroxylase exists in human liver. << Less

  FEBS Lett. 348:70-74(1994) [PubMed] [EuropePMC]

• Altered leukotriene B4 metabolism in CYP4F18-deficient mice does not impact inflammation following renal ischemia.

  Winslow V., Vaivoda R., Vasilyev A., Dombkowski D., Douaidy K., Stark C., Drake J., Guilliams E., Choudhary D., Preffer F., Stoilov I., Christmas P.

  Inflammatory responses to infection and injury must be restrained and negatively regulated to minimize damage to host tissue. One proposed mechanism involves enzymatic inactivation of the pro-inflammatory mediator leukotriene B4, but it is difficult to dissect the roles of various metabolic enzyme ... >> More

  Inflammatory responses to infection and injury must be restrained and negatively regulated to minimize damage to host tissue. One proposed mechanism involves enzymatic inactivation of the pro-inflammatory mediator leukotriene B4, but it is difficult to dissect the roles of various metabolic enzymes and pathways. A primary candidate for a regulatory pathway is omega oxidation of leukotriene B4 in neutrophils, presumptively by CYP4F3A in humans and CYP4F18 in mice. This pathway generates ω, ω-1, and ω-2 hydroxylated products of leukotriene B4, depending on species. We created mouse models targeting exons 8 and 9 of the Cyp4f18 allele that allows both conventional and conditional knockouts of Cyp4f18. Neutrophils from wild-type mice convert leukotriene B4 to 19-hydroxy leukotriene B4, and to a lesser extent 18-hydroxy leukotriene B4, whereas these products were not detected in neutrophils from conventional Cyp4f18 knockouts. A mouse model of renal ischemia-reperfusion injury was used to investigate the consequences of loss of CYP4F18 in vivo. There were no significant changes in infiltration of neutrophils and other leukocytes into kidney tissue as determined by flow cytometry and immunohistochemistry, or renal injury as assessed by histological scoring and measurement of blood urea nitrogen. It is concluded that CYP4F18 is necessary for omega oxidation of leukotriene B4 in neutrophils, and is not compensated by other CYP enzymes, but loss of this metabolic pathway is not sufficient to impact inflammation and injury following renal ischemia-reperfusion in mice. << Less

  Biochim. Biophys. Acta 1841:868-879(2014) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Human leukotriene B4 omega-hydroxylase (CYP4F3) gene: molecular cloning and chromosomal localization.

  Kikuta Y., Kato M., Yamashita Y., Miyauchi Y., Tanaka K., Kamada N., Kusunose M.

  Leukotriene B4 (LTB4) omega-hydroxylase catalyzes the conversion of LTB4 into a biologically less active product, 20-hydroxy-LTB4. In a preceding paper (Kikuta et al., 1993), we showed human polymorphonuclear leukocyte (PMN) LTB4 omega-hydroxylase to be a novel form of cytochrome P450, designated ... >> More

  Leukotriene B4 (LTB4) omega-hydroxylase catalyzes the conversion of LTB4 into a biologically less active product, 20-hydroxy-LTB4. In a preceding paper (Kikuta et al., 1993), we showed human polymorphonuclear leukocyte (PMN) LTB4 omega-hydroxylase to be a novel form of cytochrome P450, designated CYP4F3, on the basis of its cDNA cloning and expression in yeast cells. Here, we have isolated the gene encoding CYP4F3 and determined its genomic organization and chromosomal localization. The CYP4F3 gene contained 13 exons and spanned approximately 22.2 kb. The cDNA of CYP4F3 contained 5050 nucleotides excluding the poly(A) tail. The translation initiation codon (ATG) was present in exon II. Primer extension and S1 mapping analyses indicated that the transcription initiation site is 49 nucleotides upstream from the 3' end of exon I, and no other initiation sites were detected. A TATA-box-like sequence (TACAT) and 120-b GC-rich sequence were observed just before transcription initiation site. Several putative regulating elements recognized by the GATA family, MZF-1, CACCC binding protein, and C/EBP, were identified in its 5' flanking region. Genomic DNA screening for CYP4F3 and Southern blot analysis suggested the existence of other CYP4F genes in addition to CYP4F3 and CYP4F2 in the human genome. Fluorescence in situ hybridization demonstrated that the CYP4F3 gene is located at 19p13.2. << Less

  DNA Cell Biol. 17:221-230(1998) [PubMed] [EuropePMC]

• Purification and characterization of recombinant rat hepatic CYP4F1.

  Kikuta Y., Kusunose E., Ito M., Kusunose M.

  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 cont ... >> More

  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. << Less

  Arch. Biochem. Biophys. 369:193-196(1999) [PubMed] [EuropePMC]

  This publication is cited by 4 other entries.

• Functional polymorphism in human CYP4F2 decreases 20-HETE production.

  Stec D.E., Roman R.J., Flasch A., Rieder M.J.

  20-Hydroxyeicosatetraenoic acid (20-HETE) plays an important role in the regulation of renal tubular and vascular function and a deficiency in the renal formation of 20-HETE has been linked to the development of hypertension. The cytochrome P450 4F2 (CYP4F2) gene encodes for the major CYP enzyme r ... >> More

  20-Hydroxyeicosatetraenoic acid (20-HETE) plays an important role in the regulation of renal tubular and vascular function and a deficiency in the renal formation of 20-HETE has been linked to the development of hypertension. The cytochrome P450 4F2 (CYP4F2) gene encodes for the major CYP enzyme responsible for the synthesis of 20-HETE in the human kidney. We screened two human sampling panels (African and European Americans: n = 24 and 23 individuals, respectively) using PCR and DNA resequencing to identify informative SNPs in the coding region of the CYP4F2 gene. Two nonsynonymous SNPs that lead to amino acid changes at position 12 (W12G) and 433 (V433M), were identified. Both of these variants were found to be frequent in both African and European American sampling panels (9-21% minor allele frequency), and the W12G polymorphism exhibited extensive linkage disequilibrium with surrounding SNPs. To determine the functional significance of these mutations on the ability of the CYP4F2 enzyme to metabolize arachidonic acid and leukotriene B(4) (LTB(4)), recombinant baculoviruses containing four different human CYP4F2 variants (i.e., W12/V433, W12/M433, G12/V433, G12/M433) were generated and the proteins were expressed in Sf9 insect cells. The presence of the M433 allele, W12/M433, or G12/M433 decreased 20-HETE production to 56-66% of control. In contrast these variants had no effect on the omega-hydroxylation of LTB(4). These findings are the first to identify a functional variant in the human CYP4F2 gene that alters the production of 20-HETE. << Less

  Physiol. Genomics 30:74-81(2007) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Role of human CYP4F2 in hepatic catabolism of the proinflammatory agent leukotriene B4.

  Jin R., Koop D.R., Raucy J.L., Lasker J.M.

  Leukotriene B4 (LTB4), an arachidonic acid derivative, is a potent proinflammatory agent whose actions are terminated by catabolism via a microsomal omega-hydroxylation pathway. Although the liver serves as the principal site for LTB4 clearance from the systemic circulation, the attributes of hepa ... >> More

  Leukotriene B4 (LTB4), an arachidonic acid derivative, is a potent proinflammatory agent whose actions are terminated by catabolism via a microsomal omega-hydroxylation pathway. Although the liver serves as the principal site for LTB4 clearance from the systemic circulation, the attributes of hepatic LTB4 metabolism are ill defined in humans. Thus, we examined metabolism of LTB4 to its omega-hydroxylated metabolite 20-hydroxyleukotriene B4 (20-OH LTB4) by human liver microsomes and also purified the hepatic P450 enzyme underlying this reaction. Liver microsomes from 10 different subjects converted LTB4 to 20-OH LTB4 at similar rates (1.06 +/- 0.3 nmol/min/nmol P450; 0.25 +/-0.1 nmol/min/mg protein). Analysis of the microsomal LTB4 20-hydroxylation reaction revealed kinetic parameters (apparent Km of 74.8 microM with a VMAX of 2.42 nmol/min/nmol P450) consistent with catalysis by a single P450 enzyme. Conventional chromatography combined with immunochemical screening with rat CYP4A1 antibodies was then used to isolate a P450 enzyme from human liver microsomes with a molecular weight of 57,000 and an NH2-terminal amino acid sequence 94% homologous (12Trp --> 12Gly) over the first 17 residues with the human CYP4F2 cDNA-derived sequence. Upon reconstitution with P450 reductase and phospholipid, CYP4F2 converted LTB4 to 20-OH LTB4 at a turnover rate of 392 pmol/min/nmol P450, whereas the other human liver P450s tested, including CYP4A11, exhibited neglible LTB4 omega-hydroxylase activity. Polyclonal antibodies to CYP4F2 were found to markedly inhibit (91.9 +/-5%; n = 5) LTB4 20-hydroxylation by human liver microsomes. Microsomal 20-OH LTB4 formation was also inhibited 30% by arachidonic acid, a known CYP4F2 substrate, and 50% by prostaglandin A1 but was unaffected by lauric acid, palmitic acid, and PGF2alpha. Finally, a strong correlation (r = 0.86; P < 0.002; n = 10) was observed between CYP4F2 content and LTB4 20-hydroxylase activity in the human liver samples. Our results indicate that CYP4F2 is the principle LTB4 omega-hydroxylating enzyme expressed in human liver and, as such, may play an important role in regulating circulating as well as hepatic levels of this powerful proinflammatory eicosanoid. << Less

  Arch. Biochem. Biophys. 359:89-98(1998) [PubMed] [EuropePMC]

• Characterization of human liver leukotriene B(4) omega-hydroxylase P450 (CYP4F2).

  Kikuta Y., Kusunose E., Kusunose M.

  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 micr ... >> More

  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. << Less

  J. Biochem. 127:1047-1052(2000) [PubMed] [EuropePMC]

  This publication is cited by 5 other entries.

• Catalytic characterization and cytokine mediated regulation of cytochrome P450 4Fs in rat hepatocytes.

  Kalsotra A., Anakk S., Brommer C.L., Kikuta Y., Morgan E.T., Strobel H.W.

  Cytochrome P450 (CYP) 4F mediated leukotriene B(4) (LTB(4)) metabolism modulates inflammation during injury and infection. Here we show that in addition to LTB(4), the recombinant rat CYP4Fs catalyze omega-hydroxylations of lipoxin A(4), and hydroxyeicosatetraeonic acids. CYP4F gene regulation stu ... >> More

  Cytochrome P450 (CYP) 4F mediated leukotriene B(4) (LTB(4)) metabolism modulates inflammation during injury and infection. Here we show that in addition to LTB(4), the recombinant rat CYP4Fs catalyze omega-hydroxylations of lipoxin A(4), and hydroxyeicosatetraeonic acids. CYP4F gene regulation studies in primary hepatocytes reveal that pro-inflammatory cytokines interleukin (IL) -1beta, IL-6 and tumor necrosis factor (TNF) -alpha produce a general inductive response whereas IL-10, an anti-inflammatory cytokine, suppresses CYP4F expression. The molecular mechanism behind IL-6 related induction of CYP4F4 and 4F5 is partially signal transducer and activator of transcription 3 (STAT3) dependent. When hepatocytes are subjected to high concentrations of LTB(4) or prostaglandin E(2), lipid mediators of inflammation, only an increase in CYP4F5 mRNA expression is observed. Collectively, the results from isozyme activity and substrate driven CYP4F induction do not support the notion that an autoregulatory pathway could control the excessive concentrations of LTB(4) during an inflammatory challenge to hepatocytes. << Less

  Arch. Biochem. Biophys. 461:104-112(2007) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.