Takashi Iyanagi

Medical College of Wisconsin, Milwaukee, Wisconsin, United States

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Publications (41)145.49 Total impact

  • Takashi Iyanagi, Chuanwu Xia, Jung-Ja P Kim
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    ABSTRACT: NADPH-cytochrome P450 oxidoreductase (CYPOR) and nitric oxide synthase (NOS), two members of the diflavin oxidoreductase family, are multi-domain enzymes containing distinct FAD and FMN domains connected by a flexible hinge. FAD accepts a hydride ion from NADPH, and reduced FAD donates electrons to FMN, which in turn transfers electrons to the heme center of cytochrome P450 or NOS oxygenase domain. Structural analysis of CYPOR, the prototype of this enzyme family, has revealed the exact nature of the domain arrangement and the role of residues involved in cofactor binding. Recent structural and biophysical studies of CYPOR have shown that the two flavin domains undergo large domain movements during catalysis. NOS isoforms contain additional regulatory elements within the reductase domain that control electron transfer through Ca(2+)-dependent calmodulin (CaM) binding. The recent crystal structure of an iNOS Ca(2+)/CaM-FMN construct, containing the FMN domain in complex with Ca(2+)/CaM, provided structural information on the linkage between the reductase and oxgenase domains of NOS, making it possible to model the holo iNOS structure. This review summarizes recent advances in our understanding of the dynamics of domain movements during CYPOR catalysis and the role of the NOS diflavin reductase domain in the regulation of NOS isozyme activities.
    Archives of Biochemistry and Biophysics 09/2012; 528(1):72-89. · 3.37 Impact Factor
  • Chuanwu Xia, Ila Misra, Takashi Iyanagi, Jung-Ja P Kim
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    ABSTRACT: Nitric-oxide synthases (NOSs) catalyze the conversion of l-arginine to nitric oxide and citrulline. There are three NOS isozymes, each with a different physiological role: neuronal NOS, endothelial NOS, and inducible NOS (iNOS). NOSs consist of an N-terminal oxygenase domain and a C-terminal reductase domain, linked by a calmodulin (CaM)-binding region. CaM is required for NO production, but unlike other NOS isozymes, iNOS binds CaM independently of the exogenous Ca(2+) concentration. We have co-expressed CaM and the FMN domain of human iNOS, which includes the CaM-binding region. The Ca(2+)-bound protein complex (CaCaMxFMN) forms an air-stable semiquinone when reduced with NADPH and reduces cytochrome c when reconstituted with the iNOS FAD/NADPH domain. We have solved the crystal structure of the CaCaMxFMN complex in four different conformations, each with a different relative orientation, between the FMN domain and the bound CaM. The CaM-binding region together with bound CaM forms a hinge, pivots on the conserved Arg(536), and regulates electron transfer from FAD to FMN and from FMN to heme by adjusting the relative orientation and distance among the three cofactors. In addition, the relative orientations of the N- and C-terminal lobes of CaM are also different among the four conformations, suggesting that the flexibility between the two halves of CaM also contributes to the fine tuning of the orientation/distance between the redox centers. The data demonstrate a possible mode for precise control of electron transfer by altering the distance and orientation of redox centers in a protein displaying domain movement.
    Journal of Biological Chemistry 10/2009; 284(44):30708-17. · 4.65 Impact Factor
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    ABSTRACT: The liver undergoes dramatic changes in function during development. The development of UDP-glucuronosyltransferase family 1 (UGT1) isoforms was studied in livers from rats at 16-20days of gestation, at days 1, 2, 3, 4, and 7 of infancy, at days 14 and 28 of childhood, and at day 56 of young adulthood. We found developmental stage-specific switching of regulation of the rat UGT1 gene complex. UGT1A6 was expressed as a predominant component of UGT1 in fetus liver, while other UGT1 isoforms were repressed. In contrast, expression of UGT1A1 surged immediately after birth. Expression of UGT1A5 was transiently elevated in childhood. We also found age-dependent alternative usage of dual UGT1A6 promoters in rat liver. Since UGT1A1 is the only bilirubin-glucuronidating isoform, the ontogeny of UGT1A1 in liver microsomes demonstrates that inadequate UGT1A1 proteins in the early neonatal period are linked to the common etiology of idiopathic hyperbilirubinemia in the newborn infant.
    Biochemical and Biophysical Research Communications 11/2008; 377(3):815-9. · 2.41 Impact Factor
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    ABSTRACT: The object of this study was to clarify the mechanism of electron transfer in the human endothelial nitric oxide synthase (eNOS) reductase domain using recombinant eNOS reductase domains; the FAD/NADPH domain containing FAD- and NADPH-binding sites and the FAD/FMN domain containing FAD/NADPH-, FMN-, and a calmodulin-binding sites. In the presence of molecular oxygen or menadione, the reduced FAD/NADPH domain is oxidized via the neutral (blue) semiquinone (FADH(*)), which has a characteristic absorption peak at 520 nm. The FAD/NADPH and FAD/FMN domains have high activity for ferricyanide, but the FAD/FMN domain has low activity for cytochrome c. In the presence or absence of calcium/calmodulin (Ca(2+)/CaM), reduction of the oxidized flavins (FAD-FMN) and air-stable semiquinone (FAD-FMNH(*)) with NADPH occurred in at least two phases in the absorbance change at 457nm. In the presence of Ca(2+)/CaM, the reduction rate of both phases was significantly increased. In contrast, an absorbance change at 596nm gradually increased in two phases, but the rate of the fast phase was decreased by approximately 50% of that in the presence of Ca(2+)/CaM. The air-stable semiquinone form was rapidly reduced by NADPH, but a significant absorbance change at 520 nm was not observed. These findings indicate that the conversion of FADH(2)-FMNH(*) to FADH(*)-FMNH(2) is unfavorable. Reduction of the FAD moiety is activated by CaM, but the formation rate of the active intermediate, FADH(*)-FMNH(2) is extremely low. These events could cause a lowering of enzyme activity in the catalytic cycle.
    Archives of Biochemistry and Biophysics 10/2007; 465(1):254-65. · 3.37 Impact Factor
  • Takashi Iyanagi
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    ABSTRACT: Enzymes that catalyze the biotransformation of drugs and xenobiotics are generally referred to as drug-metabolizing enzymes (DMEs). DMEs can be classified into two main groups: oxidative or conjugative. The NADPH-cytochrome P450 reductase (P450R)/cytochrome P450 (P450) electron transfer systems are oxidative enzymes that mediate phase I reactions, whereas the UDP-glucuronosyltransferases (UGTs) are conjugative enzymes that mediate phase II enzymes. Both enzyme systems are localized to the endoplasmic reticulum (ER) where a number of drugs are sequentially metabolized. DMEs, including P450s and UGTs, generally have a highly plastic active site that can accommodate a wide variety of substrates. The P450 and UGT genes constitute a supergene family, in which UGT proteins are encoded by distinct genes and a complex gene. Both the P450 and UGT genes have evolved to diversify their functions. This chapter reviews advances in understanding the structure and function of the P450R/P450 and UGT enzyme systems. In particular, the coordinate biotransformation of xenobiotics by phase I and II enzymes in the ER membrane is examined.
    International Review of Cytology 02/2007; 260:35-112. · 6.09 Impact Factor
  • Takashi Iyanagi
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    ABSTRACT: NADPH-cytochrome P450 reductase (CPR) and the nitric oxide synthase (NOS) reductase domains are members of the FAD-FMN family of proteins. The FAD accepts two reducing equivalents from NADPH (dehydrogenase flavin) and FMN acts as a one-electron carrier (flavodoxin-type flavin) for the transfer from NADPH to the heme protein, in which the FMNH*/FMNH2 couple donates electrons to cytochrome P450 at constant oxidation-reduction potential. Although the interflavin electron transfer between FAD and FMN is not strictly regulated in CPR, electron transfer is activated in neuronal NOS reductase domain upon binding calmodulin (CaM), in which the CaM-bound activated form can function by a similar mechanism to that of CPR. The oxygenated form and spin state of substrate-bound cytochrome P450 in perfused rat liver are also discussed in terms of stepwise one-electron transfer from CPR. This review provides a historical perspective of the microsomal mixed-function oxidases including CPR and P450. In addition, a new model for the redox-linked conformational changes during the catalytic cycle for both CPR and NOS reductase domain is also discussed.
    Biochemical and Biophysical Research Communications 01/2006; 338(1):520-8. · 2.41 Impact Factor
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    ABSTRACT: Several novel UDP glycosyltransferase (UGT) genes, mainly UDP glucuronosyltransferases, have been identified in the human, mouse and rat genomes and in other mammalian species. This review provides an update of the UGT nomenclature to include these new genes and prevent the confusion that arises when the same gene is given different names. The new genes are named following previously established recommendations, taking into consideration evolutionary relatedness and the names already in general usage in the literature. The mammalian UGT gene superfamily currently has 117 members that can be divided into four families, UGT1, UGT2, UGT3 and UGT8. The 5-exon genes of the UGT1 family each contain a unique first exon, plus four exons that are shared between the genes; the exons 1 appear to have evolved by a process of duplication, leading to the synthesis of proteins with identical carboxyl-terminal and variable amino-terminal domains. Exon-sharing is also seen with the 6-exon UGT2A1 and UGT2A2 genes. However, UGT2A3 and those of the UGT2B (six exons), UGT3 (seven exons) and UGT8 gene families (five or six exons) do not share exons and most likely were derived by a process of duplication of all exons in the gene. Most UGT1 and UGT8 enzymes have been characterized in detail; however, the catalytic functions of the UGT3A enzymes and several UGT2 enzymes remain to be characterized.
    Pharmacogenetics and Genomics 11/2005; 15(10):677-85. · 3.61 Impact Factor
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    ABSTRACT: In cultured primary hepatocytes UDP-glucuronosyltransferase form 1A2 (UGT1A2) mRNA level is 80 times higher than that found in rat liver. We previously identified an enhancer sequence in the UGT1A2 promoter, and designated it as culture-associated expression responsive enhancer module (CEREM). Affinity chromatography with DNA fragments containing CEREM allowed enrichment of nuclear factor I (NFI) proteins from cultured hepatocytes. The NFI family is encoded by four distinct genes, NFI-A, NFI-B, NFI-C, and NFI-X. Immunoblot analysis with isoform-specific antibodies showed that NFI-A1 existed as a major component in rat liver and cultured hepatocytes. By contrast, NFI-C1 was present in rat liver but disappeared immediately upon cultivation of hepatocytes. Only trace amounts of NFI-B and NFI-X were detectable in rat liver and cultured hepatocytes. NFI-A1 elevated expression of the reporter gene that is under the control of CEREM, while NFI-C1 had an inhibitory effect. Co-expression of a constant amount of NFI-A1 with an increasing amount of NFI-C1 led to a concentration-dependent decrease in the expression of the CEREM-controlled reporter gene mediated by NFI-A1. Activation of UGT1A2 expression by NFI-A1 is suppressed by the coexistence of NFI-C1 in the liver, and culture-associated expression of UGT1A2 is triggered by the rapid disappearance of NFI-C1 in cultured hepatocytes.
    Journal of Biochemistry 10/2005; 138(3):313-25. · 3.07 Impact Factor
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    ABSTRACT: In this study, we have analyzed interflavin electron transfer reactions from FAD to FMN in both the full-length inducible nitric oxide synthase (iNOS) and its reductase domain. Comparison is made with the interflavin electron transfer in NADPH-cytochrome P450 reductase (CPR). For the analysis of interflavin electron transfer and the flavin intermediates observed during catalysis we have used menadione (MD), which can accept an electron from both the FAD and FMN sites of the enzyme. A characteristic absorption peak at 630 and 520 nm can identify each FAD and FMN semiquinone species, which is derived from CPR and iNOS, respectively. The charge transfer complexes of FAD with NADP+ or NADPH were monitored at 750 nm. In the presence of MD, the air-stable neutral (blue) semiquinone form (FAD-FMNH*) was observed as a major intermediate during the catalytic cycle in both the iNOS reductase domain and full-length enzyme, and its formation occurred without any lag phase indicating rapid interflavin electron transfer following the reduction of FAD by NADPH. These data also strongly suggest that the low level reactivity of a neutral (blue) FMN semiquinone radical with electron acceptors enables one-electron transfer in the catalytic cycle of both the FAD-FMN pairs in CPR and iNOS. On the basis of these data, we propose a common model for the catalytic cycle of both CaM-bound iNOS reductase domain and CPR.
    Archives of Biochemistry and Biophysics 09/2005; 440(1):65-78. · 3.37 Impact Factor
  • Shigenobu Kimura, Tomoka Umemura, Takashi Iyanagi
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    ABSTRACT: Two-cistronic expression plasmids for the wild-type solubilized domain of porcine NADPH-cytochrome P450 reductase (PsCPR) gene in Escherichia coli were systematically constructed using a solubilized domain of porcine cytochrome b5 gene (Psb5 gene) or a derivative of it as the first cistron to examine their utility for second gene expression preventing the translational inhibition caused by the intramolecular local secondary structure of mRNA at the ribosome-binding site (RBS). The mRNAs from the plasmids lacking an RBS for the second cistron (SD2) accumulated very low levels of PsCPR, while those from the plasmids having an SD2 accumulated higher levels of PsCPR. The level of accumulation of PsCPR by the mRNA from plasmid pCbSD-T-CPR-3, which has an SD2 upstream of the termination codon of the first cistron, was higher than for those with an SD2 in the intercistronic region. The predicted intramolecular local secondary structures at the SD2 of mRNAs from these plasmids were stable enough to cause translational initiation inhibition. These results indicate that the use of a two-cistronic expression plasmid is an effective way to overcome translational initiation inhibition. Improved plasmids, pCP1 and pCP2P, were constructed from pCbSD-T-CPR-3. Using these plasmids, the solubilized donain of porcine NADH-cytochrome b5 reductase was also highly accumulated on prevention of the translational initiation inhibition. These plasmids are expected to be useful tools for the comprehensive high-level expression of heterologous genes in E. coli cells.
    Journal of Biochemistry 05/2005; 137(4):523-33. · 3.07 Impact Factor
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    ABSTRACT: 26,26,26,27,27,27-Hexafluoro-1alpha,25-dihydroxyvitamin D(3) [F(6)-1alpha, 25(OH)(2)D(3)], which is now clinically used as a drug for secondary hyperparathyroidism, is a hexafluorinated analog of the active form of vitamin D(3). Our previous studies demonstrated that CYP24A1 is responsible for the metabolism of F(6)-1alpha,25(OH)(2)D(3) in the target tissues and that F(6)-1alpha,25(OH)(2)D(3) was successively converted to F(6)-1alpha,23S,25(OH)(3)D(3) and F(6)-23-oxo-1alpha,25(OH)(2)D(3). In this study, we examined the metabolism of F(6)-1alpha,25(OH)(2)D(3),F(6)-1alpha,23S,25(OH)(3)D(3), and F(6)-23-oxo-1alpha,25(OH)(2)D(3) by human UDP-glucuronosyltransferases (UGTs). Of these compounds, F(6)-1alpha,23S,25(OH)(3)D(3) was remarkably glucuronidated both in human liver microsomes and in the recombinant system expressing human UGT. No significant interindividual differences were observed among 10 human liver samples. The recombinant system for 12 species of human UGTs revealed that F(6)-1alpha,23S,25(OH)(3)D(3) glucuronidation was specifically catalyzed by UGT1A3. The information obtained in this study seems very useful to predict the metabolism and efficacy of vitamin D analogs in human bodies before clinical trials. In addition, note that for the first time a possible probe substrate for UGT1A3 has been found.
    Drug Metabolism and Disposition 02/2005; 33(1):102-7. · 3.36 Impact Factor
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    ABSTRACT: Metabolism of polychlorinated dibenzo-p-dioxins by cytochrome P450 (P450) and UDP-glucuronosyltransferase (UGT) was examined using a recombinant enzyme system and human liver microsomes. We analyzed the glucuronidation of 2,3,7-trichlorodibenzo-p-dioxin (2,3,7-triCDD) by rat CYP1A1 expressed in yeast microsomes and human UGT expressed in baculovirus-infected insect cells. Multiple UGT isozymes showed glucuronidation activity toward 8-hydroxy-2,3,7-triCDD (8-OH-2,3,7-triCDD), which was produced by CYP1A1. Of these UGTs, UGT1A1, 1A9, and 2B7, which are constitutively expressed in human livers, showed remarkable activity toward 8-OH-2,3,7-triCDD. The apparent kinetic parameters of glucuronidation, K(m) and k(cat), were estimated to be 0.8 microM and 1.8 min(-1), respectively, for UGT1A1, 0.8 microM and 1.8 min(-1), respectively, for UGT1A9, and 3.9 microM and 7.0 min(-1), respectively, for UGT2B7. In human liver microsomes with NADPH and UDP-glucuronic acid, 2,3,7-triCDD was first converted to 8-OH-2,3,7-triCDD, then further converted to its glucuronide. We compared the ability of 10 human liver microsomes to metabolize 2,3,7-triCDD and observed a significant difference in the glucuronidation of 2,3,7-triCDD that originated from the difference of the P450-dependent hydroxylation of 2,3,7-triCDD.
    Drug Metabolism and Disposition 09/2004; 32(8):870-5. · 3.36 Impact Factor
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    ABSTRACT: We have analyzed the mechanism of one-electron reduction of adriamycin (Adr) using recombinant full-length human neuronal nitric-oxide synthase and its flavin domains. Both enzymes catalyzed aerobic NADPH oxidation in the presence of Adr. Calcium/calmodulin (Ca(2+)/CaM) stimulated the NADPH oxidation of Adr. In the presence or absence of Ca(2+)/CaM, the flavin semiquinone radical species were major intermediates observed during the oxidation of the reduced enzyme by Adr. The FAD-NADPH binding domain did not significantly catalyze the reduction of Adr. Neither the FAD semiquinone (FADH*) nor the air-stable semiquinone (FAD-FMNH*) reacted rapidly with Adr. These data indicate that the fully reduced species of FMN (FMNH(2)) donates one electron to Adr, and that the rate of Adr reduction is stimulated by a rapid electron exchange between the two flavins in the presence of Ca(2+)/CaM. Based on these findings, we propose a role for the FAD-FMN pair in the one-electron reduction of Adr.
    Archives of Biochemistry and Biophysics 08/2004; 427(2):180-7. · 3.37 Impact Factor
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    ABSTRACT: Xenobiotic Phase I and Phase II reactions in hepatocytes occur sequentially and cooperatively during the metabolism of various chemical compounds including drugs. In order to investigate the sequential metabolism of 7-ethoxycoumarin (7EC) as model substrate in vitro, xenobiotic metabolizing enzymes, rat cytochrome P450 1A1 (P450 1A1) and UDP-glucuronosyltransferase 1A6 (UGT1A6) were co-expressed in Saccharomyces cerevisiae AH22. Rat P450 1A1 and yeast NADPH-P450 reductase were expressed on a multicopy plasmid (pGYR1) in the yeast. Rat UGT1A6 cDNA with a yeast alcohol dehydrogenase I promoter and terminator was integrated into yeast chromosomal DNA to achieve the stable expression. Co-expression of P450 1A1 and UGT1A6 in yeast microsomes was confirmed by immunoblot analysis. Protease treatment of the microsomes showed the correct topological orientation of UGT to the membranes. The metabolism of 7EC to 7-hydroxycoumarin (7HC) and its glucuronide in yeast microsomes was analyzed by reverse phase HPLC. In a co-expression system containing 7EC, NADPH and UDP-glucuronic acid, glucuronide formation was detected after a lag phase, following the accumulation of 7HC. In the case of P450 1A1 and UGT1A6, efficient coupling of hydroxylation and glucuronidation in 7EC metabolism was not observed in the co-expression system. This P450 and UGT co-expression system in yeast allows the sequential biotransformation of xenobiotics to be simulated in vitro.
    Biochimica et Biophysica Acta 06/2004; 1672(2):86-92. · 4.66 Impact Factor
  • Shigenobu Kimura, Takashi Iyanagi
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    ABSTRACT: A direct expression system for the solubilized catalytic domains of NADPH-cytochrome P-450 reductase (sCPR) from rat (RsCPR) and porcine (PsCPR) in Escherichia coli cells was constructed using the expression plasmid pCWori(+). PsCPR was minimally expressed, whereas RsCPR was highly expressed. Replacement of the nucleotides encoding Thr(60)Ser(61)Ser(62) in PsCPR with those for Ala(60)Pro(61)Pro(62) in RsCPR markedly increased the expression level of the protein. The local secondary structures of the mRNAs, which were predicted with the prediction program GeneBee (http://www.genebee.msu.su), suggested that the intramolecular double strand between the ribosome binding site (RBS) and the Thr(60)Ser(61)Ser(62) codons in PsCPR, and/or the base-pairing at the initiation codon of the mRNAs significantly affected protein expression. Silent mutations were systematically introduced into the codons for Thr(58) and Thr(60)Ser(61) in PsCPR to modulate the local secondary structure of the mRNA. The expression level of the silently mutated PsCPR suggests that the expression level of PsCPR depends on the stability of the local structure at the RBS in the mRNA. A high-level expression system for wild-type PsCPR was constructed by introducing silent mutations at the codons for Thr(60)Ser(61) in PsCPR. The purified PsCPR showed the characteristic absorption spectral changes of sCPR after reduction with NADPH. The yield of purified PsCPR from 1 liter of culture fluid was 45.8 mg. These results substantiate that the introduction of silent mutations in the section of the gene encoding the N-terminal region of the protein based on the predicted local secondary structure of the mRNA at the RBS is a useful approach to control and increase the expression level of heterologous proteins in E. coli cells.
    Journal of Biochemistry 10/2003; 134(3):403-13. · 3.07 Impact Factor
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    ABSTRACT: Neuronal nitric-oxide synthase (nNOS) differs from inducible NOS (iNOS) in both its dependence on the intracellular Ca2+ concentration and the production rate of NO. To investigate what difference(s) exist between the two NOS flavin domains at the electron transfer level, we isolated the recombinant human NOS flavin domains, which were co-expressed with human calmodulin (CaM). The flavin semiquinones, FADH* and FMNH*, in both NOSs participate in the regulation of one-electron transfer within the flavin domain. Each semiquinone can be identified by a characteristic absorption peak at 520 nm (Guan, Z.-W., and Iyanagi, T. (2003) Arch. Biochem. Biophys. 412, 65-76). NADPH reduction of the FAD and FMN redox centers by the CaM-bound flavin domains was studied by stopped-flow and rapid scan spectrometry. Reduction of the air-stable semiquinone (FAD-FMNH*) of both domains with NADPH showed that the extent of conversion of FADH2/FMNH* to FADH*/FMNH2 in the iNOS flavin domain was greater than that of the nNOS flavin domain. The reduction of both oxidized domains (FAD-FMN) with NADPH resulted in the initial formation of a small amount of disemiquinone, which then decayed. The rate of intramolecular electron transfer between the two flavins in the iNOS flavin domain was faster than that of the nNOS flavin domain. In addition, the formation of a mixture of the two- and four-electron-reduced states in the presence of excess NADPH was different for the two NOS flavin domains. The data indicate a more favorable formation of the active intermediate FMNH2 in the iNOS flavin domain.
    Journal of Biological Chemistry 09/2003; 278(33):30859-68. · 4.65 Impact Factor
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    ABSTRACT: Spironolactone (SL) increases the glucuronidation rate of several compounds. We analyzed the molecular basis of changes occurring in major rat liver UDP-glucuronosyltransferase (UGT) family 1 isoforms and in UGT2B1, a relevant isoform of family 2, in response to SL. UGT activity toward bilirubin, ethynylestradiol and p-nitrophenol was assayed in native and activated microsomes. Protein and mRNA levels were determined by Western and Northern blotting. The lipid composition and physicochemical properties of the microsomal membrane were also analyzed. Glucuronidation rates of bilirubin and ethynylestradiol (at both 3-OH and 17 beta-OH positions), determined in UDP-N-acetylglucosamine-activated membranes, were increased in SL group. Western blot analysis revealed increased levels of UGT1A1 and 1A5 (bilirubin and 3-OH ethynylestradiol conjugation), and 2B1 (17 beta-OH ethynylestradiol conjugation). Northern blot studies suggested transcriptional regulation by the steroid. Analysis of UGT activity in native vs. alamethicin-activated microsomes indicated increased latency, which was not associated to changes in physicochemical properties of the microsomal membrane. p-Nitrophenol glucuronidation rate and mRNA and protein levels of UGT1A6, the main isoform conjugating planar phenols, were not affected by the inducer. The data suggest transcriptional regulation of specific isoforms of hepatic UGT by SL, thus explaining previously reported increases in UGT activity toward selective substrates.
    Biochemical Pharmacology 08/2003; 66(1):171-7. · 4.58 Impact Factor
  • Zhi-Wen Guan, Takashi Iyanagi
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    ABSTRACT: The objective of this study was to clarify the mechanism of electron transfer in the human neuronal nitric oxide synthase (nNOS) flavin domain using the recombinant human nNOS flavin domains, the FAD/NADPH domain (contains FAD- and NADPH-binding sites), and the FAD/FMN domain (the flavin domain including a calmodulin-binding site). The reduction by NADPH of the two domains was studied by rapid-mixing, stopped-flow spectroscopy. For the FAD/NADPH domain, the results indicate that FAD is reduced by NADPH to generate the two-electron-reduced form (FADH(2)) and the reoxidation of the reduced FAD proceeds via a neutral (blue) semiquinone with molecular oxygen or ferricyanide, indicating that the reduced FAD is oxidized in two successive one-electron steps. The neutral (blue) semiquinone form, as an intermediate in the air-oxidation, was unstable in the presence of O(2). The purified FAD/NADPH domain prepared under our experimental conditions was activated by NADP(+) but not NAD(+). These results indicate that this domain exists in two states; an active state and a resting state, and the enzyme in the resting state can be activated by NADP(+). For the FAD/FMN domain, the reduction of the FAD-FMN pair of the oxidized enzyme with NADPH proceeded by both one-electron equivalent and two-electron equivalent mechanisms. The formation of semiquinones from the FAD-FMN pair was greatly increased in the presence of Ca(2+)/CaM. The air-stable semiquinone form, FAD-FMNH(.), was further rapidly reduced by NADPH with an increase at 520 nm, which is a characteristic peak of the FAD semiquinone. Results presented here indicate that intramolecular one-electron transfer from FAD to FMN is activated by the binding of Ca(2+)/CaM.
    Archives of Biochemistry and Biophysics 05/2003; 412(1):65-76. · 3.37 Impact Factor
  • Takashi Iyanagi
    Seikagaku. The Journal of Japanese Biochemical Society 04/2003; 75(3):234-41. · 0.04 Impact Factor
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    ABSTRACT: Sensitive capillary electrophoresis (CE) methods are required for emerging areas of biochemical research such as the metabolome. In this report, dynamic pH junction-sweeping CE with laser-induced fluorescence (LIF) detection is applied as a robust single method to analyze trace amounts of three flavin derivatives, riboflavin, flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD), from several types of samples including bacterial cell extracts, recombinant protein, and biological fluids. Submicromolar amounts of flavin coenzymes were measured directly from formic acid cell extracts of Bacillus subtilis. Significant differences in flavin concentration were measured in cell extracts derived from either glucose or malate as the carbon source in the culture media. Quantitative assessment of FAD and FMN content from selected flavoenzymes was demonstrated after heat denaturation to release noncovalently bound coenzymes and deproteinization. This method was also applied to the analysis of free flavins in pooled human plasma and urine without the need for laborious off-line sample preconcentration. Picomolar detectability of flavins by CE-LIF detection was realized with on-line preconcentration (up to 15% capillary length used for injection) by dynamic pH junction-sweeping, resulting in a limit of detection (S/N = 3) of about 4.0 pM for FAD and FMN. This represents over a 60-fold improvement in concentration sensitivity compared to those of previous techniques using conventional injections. The method was validated in terms of reproducibility, sensitivity, linearity, and specificity. Flavin analysis by dynamic pH junction-sweeping CE-LIF offers a simple, yet sensitive way to analyze trace levels of flavin metabolites from complex biological samples.
    Analytical Biochemistry 03/2003; 313(1):89-96. · 2.58 Impact Factor

Publication Stats

1k Citations
145.49 Total Impact Points


  • 2012
    • Medical College of Wisconsin
      • Department of Biochemistry
      Milwaukee, Wisconsin, United States
  • 1996–2012
    • Himeji Institute of Technology
      • • Graduate School of Science
      • • Faculty of Science
      • • Department of Life Science
      Himezi, Hyōgo, Japan
  • 2005–2008
    • University of Hyogo
      • Graduate School of Life Science
      Akō, Hyogo-ken, Japan
  • 2006
    • SPring-8
      Saitama, Saitama, Japan