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Cytochrome P450 Reductase (cytochrome + p450_reductase)
Selected AbstractsThermodynamic and kinetic analysis of the isolated FAD domain of rat neuronal nitric oxide synthase altered in the region of the FAD shielding residue Phe1395FEBS JOURNAL, Issue 12 2004Adrian J. Dunford In rat neuronal nitric oxide synthase, Phe1395 is positioned over the FAD isoalloxazine ring. This is replaced by Trp676 in human cytochrome P450 reductase, a tryptophan in related diflavin reductases (e.g. methionine synthase reductase and novel reductase 1), and tyrosine in plant ferredoxin-NADP+ reductase. Trp676 in human cytochrome P450 reductase is conformationally mobile, and plays a key role in enzyme reduction. Mutagenesis of Trp676 to alanine results in a functional NADH-dependent reductase. Herein, we describe studies of rat neuronal nitric oxide synthase FAD domains, in which the aromatic shielding residue Phe1395 is replaced by tryptophan, alanine and serine. In steady-state assays the F1395A and F1395S domains have a greater preference for NADH compared with F1395W and wild-type. Stopped-flow studies indicate flavin reduction by NADH is significantly faster with F1395S and F1395A domains, suggesting that this contributes to altered preference in coenzyme specificity. Unlike cytochrome P450 reductase, the switch in coenzyme specificity is not attributed to differential binding of NADPH and NADH, but probably results from improved geometry for hydride transfer in the F1395S, and F1395A,NADH complexes. Potentiometry indicates that the substitutions do not significantly perturb thermodynamic properties of the FAD, although considerable changes in electronic absorption properties are observed in oxidized F1395A and F1395S, consistent with changes in hydrophobicity of the flavin environment. In wild-type and F1395W FAD domains, prolonged incubation with NADPH results in development of the neutral blue semiquinone FAD species. This reaction is suppressed in the mutant FAD domains lacking the shielding aromatic residue. [source] Interflavin electron transfer in human cytochrome P450 reductase is enhanced by coenzyme bindingFEBS JOURNAL, Issue 12 2003Relaxation kinetic studies with coenzyme analogues The role of coenzyme binding in regulating interflavin electron transfer in human cytochrome P450 reductase (CPR) has been studied using temperature-jump spectroscopy. Previous studies [Gutierrez, A., Paine, M., Wolf, C.R., Scrutton, N.S., & Roberts, G.C.K. Biochemistry (2002) 41, 4626,4637] have shown that the observed rate, 1/,, of interflavin electron transfer (FADsq , FMNsq,FADox , FMNhq) in CPR reduced at the two-electron level with NADPH is 55 ± 2 s,1, whereas with dithionite-reduced enzyme the observed rate is 11 ± 0.5 s,1, suggesting that NADPH (or NADP+) binding has an important role in controlling the rate of internal electron transfer. In relaxation experiments performed with CPR reduced at the two-electron level with NADH, the observed rate of internal electron transfer (1/, = 18 ± 0.7 s,1) is intermediate in value between those seen with dithionite-reduced and NADPH-reduced enzyme, indicating that the presence of the 2,-phosphate is important for enhancing internal electron transfer. To investigate this further, temperature jump experiments were performed with dithionite-reduced enzyme in the presence of 2,,5,-ADP and 2,-AMP. These two ligands increase the observed rate of interflavin electron transfer in two-electron reduced CPR from 1/, = 11 s,1 to 35 ± 0.2 s,1 and 32 ± 0.6 s,1, respectively. Reduction of CPR at the two-electron level by NADPH, NADH or dithionite generates the same spectral species, consistent with an electron distribution that is equivalent regardless of reductant at the initiation of the temperature jump. Spectroelectrochemical experiments establish that the redox potentials of the flavins of CPR are unchanged on binding 2,,5,-ADP, supporting the view that enhanced rates of interdomain electron transfer have their origin in a conformational change produced by binding NADPH or its fragments. Addition of 2,,5,-ADP either to the isolated FAD-domain or to full-length CPR (in their oxidized and reduced forms) leads to perturbation of the optical spectra of both the flavins, consistent with a conformational change that alters the environment of these redox cofactors. The binding of 2,,5,-ADP eliminates the unusual dependence of the observed flavin reduction rate on NADPH concentration (i.e. enhanced at low coenzyme concentration) observed in stopped-flow studies. The data are discussed in the context of previous kinetic studies and of the crystallographic structure of rat CPR. [source] Mechanism of inhibition of purified leaping mullet (liza saliens) NADPH-cytochrome P450 reductase by toxic metals: Aluminum and thalliumJOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 6 2007Azra Bozcaarmutlu Abstract Aluminum and thallium may reach life-threatening levels in aquatic systems in the near future because of their extensive use in various industrial fields. It is therefore important to study the mechanism of toxicity of aluminum and thallium on fish enzymes. To this aim, the effects of aluminum and thallium on the activity of purified leaping mullet (Liza saliens) cytochrome P450 reductase, an essential component of the important cytochrome P450 system, have been studied. Results indicated that both metal ions strongly inhibited the NADPH-cytochrome P450 reductase. The IC50 values of AlCl3 and TlCl3 were estimated to be 34 ,M and 3 ,M, respectively. The Lineweaver,Burk plot and Dixon plot revealed that both metal ions noncompetitively inhibited the purified mullet cytochrome P450 reductase. The Ki values of Al3+ and Tl3+ were calculated from Dixon plots as 8.9 and 5.6 ,M, respectively. The inhibitory effects of Al3+ and Tl3+ on purified cytochrome P450 reductase were partially recovered by 1 mM EDTA. Additionally, tin and magnesium were shown to have no apparent effect on purified mullet cytochrome P450 reductase. © 2007 Wiley Periodicals, Inc. J Biochem Mol Toxicol 21:340,3347, 2007; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20200 [source] Biochemical characteristics of purified beef liver NADPH,cytochrome P450 reductaseJOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 6 2002Emel Arinç Abstract NADPH,cytochrome P450 reductase, an obligatory component of the cytochrome P450 dependent monooxygenase system, was purified to electrophoretic homogeneity from beef liver microsomes. The purification procedure involved the ion exchange chromatography of the detergent-solubilized microsomes on first and second DEAE-cellulose columns, followed by 2,,5,-ADP Sepharose affinity chromatography. Further concentration of the enzyme and removal of Emulgen 913 and 2,-AMP were accomplished on the final hydroxylapatite column. The enzyme was purified 239-fold and the yield was 13.5%. Monomer molecular weight of the enzyme was estimated to be 76000 ± 3000 (N = 5) by SDS-PAGE. The absolute absorption spectrum of beef reductase showed two peaks at 455 and 378 nm, with a shoulder at 478 nm, characteristics of flavoproteins. The effects of cytochrome c concentration, pH, and ionic strength on enzyme activity were studied. Reduction of cytochrome c with the enzyme followed Michaelis,Menten kinetics, and the apparent Km of the purified enzyme was found to be 47.7 ,M for cytochrome c when the enzyme activity was measured in 0.3 M potassium phosphate buffer (pH 7.7). Stability of cytochrome c reductase activity was examined at 25 and 37°C in the presence and absence of 20% glycerol. The presence of glycerol enhanced the stability of cytochrome c reductase activity at both temperatures. Sheep lung microsomal cytochrome P4502B and NADPH,cytochrome P450 reductase were also purified by the already existing methods developed in our laboratory. Both beef liver and sheep lung reductases were found to be effective in supporting benzphetamine and cocaine N-demethylation reactions in the reconstituted systems containing purified sheep lung cytochrome P4502B and synthetic lipid, phosphatidylcholine dilauroyl. © 2002 Wiley Periodicals, Inc. J Biochem Mol Toxicol 16:286,297, 2002; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.10054 [source] Modulatory influence of Andrographis paniculata on mouse hepatic and extrahepatic carcinogen metabolizing enzymes and antioxidant statusPHYTOTHERAPY RESEARCH, Issue 5 2001Rana P. Singh Abstract The effects of two doses (50 and 100,mg/kg body wt/day for 14 days) of an 80% hydroalcohol extract of Andrographis paniculata and butylated hydroxyanisole (BHA) were examined on drug metabolizing enzymes, antioxidant enzymes, glutathione content, lactate dehydrogenase (LDH) and lipid peroxidation in the liver of Swiss albino mice (6,8 weeks old). The effect of the extract and BHA were also examined on lung, kidney and forestomach for the activities of glutathione S-transferase (GST), DT-diaphorase (DTD), superoxide dismutase (SOD) and catalase. A significant increase in the levels of acid soluble sulphydryl (-SH) content, cytochrome P450, cytochrome P450 reductase, cytochrome b5 reductase, GST, DTD and SOD were observed at both dose levels of extract treatment while catalase, glutathione peroxidase and glutathione reductase (GR) showed significant increases only at the higher dose in the liver. Both Andrographis treated groups showed a significant decrease in activity of LDH and malondialdehyde (MDA) formation. BHA treated mice showed a significant increase in the levels of cytochrome b5, GST, DTD, -SH content, GR and catalase in liver; while LDH and MDA levels were reduced significantly compared with their control values. In the lung, SOD, catalase and DTD, in the kidney catalase, DTD and GST, and in the forestomach SOD and DTD showed a significant increase at both dose levels of treatment. In BHA treated mice GST, DTD and catalase were significantly induced in the lung and along with these enzymes SOD was also induced in the kidney. In the case of the forestomach of BHA treated mice GST, DTD and SOD were enhanced significantly. These findings indicate the chemopreventive potential of Andrographis paniculata against chemotoxicity including carcinogenicity. Copyright © 2001 John Wiley & Sons, Ltd. [source] Localization of Pyruvate:NADP+ Oxidoreductase in Sporozoites of Cryptosporidium parvumTHE JOURNAL OF EUKARYOTIC MICROBIOLOGY, Issue 4 2006VLASTA CTRNACTA ABSTRACT. Cryptosporidium parvum contains a unique fusion protein pyruvate:NADP+ oxidoreductase (CpPNO) that is composed of two distinct, conserved domains, an N-terminal pyruvate:ferredoxin oxidoreductase (PFO) and a C-terminal cytochrome P450 reductase (CPR). Unlike a similar fusion protein that localizes to the mitochondrion of the photosynthetic protist Euglena gracilis, CpPNO lacks an N-terminal mitochondrial targeting sequence. Using two distinct polyclonal antibodies raised against CpPFO and one polyclonal antibody against CpCPR, Western blot analysis has shown that sporozoites of C. parvum express the entire CpPNO fusion protein. Furthermore, confocal immunofluorescence and transmission electron microscopy confirm that CpPNO is localized within the cytosol rather than the relict mitochondrion of C. parvum. The distribution of this protein is not, however, strictly confined to the cytosol. CpPNO also appears to localize posteriorly within the crystalloid body. [source] Initiation of Steroidogenesis Precedes Expression of Cholesterologenic Enzymes in the Fetal Mouse TestesANATOMIA, HISTOLOGIA, EMBRYOLOGIA, Issue 6 2009T. Büdefeld Summary Sexual differentiation is a carefully regulated process that ultimately results in a development of the male or female phenotype. Proper development of the male phenotype is dependent upon the action of testosterone and anti-mullerian hormone. Leydig cells start to produce testosterone around day 12.5 in the fetal mouse testis, and continue to produce high levels of this hormone throughout gestation. In the present study, we examined whether expression of lanosterol 14,-demethylase (cyp51) and cytochrome P450 NADPH reductase, both involved in the cholesterol production, occurs simultaneously with proteins required for the production of steroid hormones. Immunocytochemical staining with the antibodies against cyp51, cytochrome P450 NADPH reductase, steroidogenic acute regulatory protein (StAR) and 3beta-hydroxysteroid dehydrogenase I (3,-HSD I) was used to determine the ontogeny of expression of these four proteins. As expected, 3,-HSD I and StAR proteins were detected on day 12.5 p.c., while expression of cyp51 and NADPH cytochrome P450 reductase appeared 1 day later, on day 13.5. Thereafter, the expression of all four proteins remained strong throughout gestation. Results of this study suggest that initial steps of steroid hormone production in murine Leydig cells are mostly dependent on exogenously derived cholesterol, while from day 13.5 onwards, mouse Leydig cells are able to synthesize cholesterol and are therefore not dependent on exogenous cholesterol resources. [source] | |||||||||||||