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经苯巴比妥钠诱导的雄性大白鼠的肝微粒体纯化的细胞色素P-450同功酶组份,经SDS-PAGE鉴定呈电泳纯,分子量为55kD。部分纯化的NADPH-细胞色素P-450还原酶,含72和77kD两个蛋白质组分。上述细胞色素P-450和NADPH-细胞色素P-450还原酶与卵磷脂制备的脂质体重组后的活性试验表明,对艾氏剂有环氧化作用,对环已烷有羟化作用,对溴氰菊酯的羟化作用微弱。当重组系统中缺少细胞色素P-450组份时,对环已烷不再起作用。同时还研究了纯化的细胞色素P-450的光谱特性。 相似文献
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云芝Trametes versicolor具有很强的环境有机污染物降解能力,其烟酰胺腺嘌呤二核苷酸-细胞色素P450还原酶(NADPH-cytochrome P450 reductase,CPR)为细胞色素P450酶(Cytochrome P450s,CYPs)提供电子,参与有机污染物的降解过程。序列分析显示,云芝基因组拥有1个潜在CPR序列和多个潜在CYP序列。为深入研究云芝CPR参与细胞降解有机污染物的分子机制,实验进行了云芝CPR在大肠杆菌中异源表达和酶学特性分析。结果表明,经IPTG诱导后,去除预测的N端膜锚定区域(氨基酸残基1–24)的CPR蛋白(CPRΔ24)可在重组菌中实现可溶性表达,且表达蛋白的分子量与理论值(78 kDa)一致。镍离子亲和层析和分子筛层析纯化后测得其比活性为5.82 U/mg。酶学性质分析显示,重组CPRΔ24的最适温度和pH分别为35℃和8.0,并对一些金属离子及有机溶剂具有不同程度的耐受性。酶在35℃、pH 8.0反应条件下对NADPH的动力学参数K_m和k_(cat)分别为19.7μmol/L、3.31/s;对底物细胞色素c的动力学参数K_m和k_(cat)分别为25.9μmol/L、10.2/s。以上研究为探究云芝CPR在环境有机污染物降解途径中的功能机制奠定基础。 相似文献
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10-羟基-2-癸烯酸(10-HDA)是蜂王浆中的主要脂肪酸成分,具有抗菌、抗癌、延缓衰老等多种生理活性,但目前关于10-HDA生物合成的分子机制还不清楚。通过克隆蜜蜂NADPH-细胞色素P450还原酶(EC 1.6.2.4,NADPH-cytochrome P450 reductase,CPR),在大肠杆菌中异源表达,并对其酶学特性进行分析。结果表明重组菌经IPTG诱导后表达蛋白的分子量与预期一致,为86.29 kDa,Ni-NTA亲和纯化后测得其比活性为77.33(EU of CPR)/μg。酶学性质分析结果表明蜜蜂CPR酶最适温度与pH分别为40℃和8.0,并对一些金属离子及有机溶剂具有不同程度的耐受性。其对底物细胞色素C的动力学参数Km和kcat分别为76 μM和268/min。以上研究为探究CPR在10-HDA生物合成途径中的功能奠定理论基础。 相似文献
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分离提纯的辅酶Q细胞色素c还原酶经胆酸盐透析法重组于各种脂质体上,发现脂质体中PE含量的升高可显著提高辅酶Q-细胞色素c还原酶的呼吸控制率.在PE含量为60%-80%时达到最高值,DOPE的L_x—H_Ⅱ的相变温度显著低于DEPE,脂酶体中DOPE含量的提高,可显著提高酶的呼吸控制率,而DEPE的影响很小,改变脂酶体中心磷脂的含量对呼吸控制率的影响较小.含有PE脂酶体中酶的CD谱419nm处的正峰,随PE含量的增大而增强,表明酶蛋白中血红素辅基的微环境有所改变.NBD-DOPE标记脂酶体研究了不同脂酶体的 L_x-H_Ⅱ的相变性质,表明PE含量的降低脂质体的相变温度上升甚至消失. 相似文献
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琥珀酸细胞色素c还原酶除去90%以上的磷脂后活力丧失约95%。将去脂琥珀酸细胞色素c还原酶与磷脂和辅酶Q_2保温,可恢复其活性。活力恢复程度依赖于磷脂的组成。当磷脂酰胆碱(PC):心磷脂(CL):磷脂酰乙醇胺(PE)=2:2:1时活力恢复最高,比大豆磷脂的效果更为明显,单组分PC,PE或CL恢复活力较差。与酶蛋白紧密结合的CL和PC在活力可逆恢复中有重要作用。 相似文献
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用羟基磷灰石柱亲和层析法制备了高纯度的缺脂泛醌细胞色素c还原酶.脂的缺失使该酶活力丢失,部分细胞色素(约52.8%细胞色素b和82.5%细胞色素c1)呈现还原状态.将缺脂泛醌细胞色素。还原酶与磷脂重组,可恢复其活性,同时那些呈还原状态的细胞色素也恢复到氧化态.此结果表明如此制备的缺脂泛醌细胞色素c还原酶仍保持着活力所必需的构象状态,细胞色素氧化还原状态随脂缺失的变化反映了脂与蛋白的相互作用. 相似文献
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NADH-细胞色素 b5 还原酶在甲状腺过氧化氢生物合成中的作用 总被引:1,自引:0,他引:1
应用高香草酸荧光分析技术及NADH-高铁氰化钾还原酶法,对正常和Graves病甲状腺过氧化氢(H2O2)和NADH-细胞色素b5还原酶(b5R)进行测定,发现Graves病甲状腺b5R活性和H2O2水平均明显高于正常,而H2O2酶活性在Graves病和正常甲状腺间无显著差异。加b5R抑制剂对氯汞苯甲酸抑制b5R活性,Graves病和正常甲状腺b5R活性降低近85%,同时H2O2降低近50%,蛋白结合碘形成减少近52%。b5R活性和H2O2水平两者呈显著正相关关系。以上结果表明,b5R参与甲状腺内H2O2的生物合成,是甲状腺内产生H2O2的重要酶系。 相似文献
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猪肝微粒体NADH—细胞色素b5还原酶的纯化及特性分析 总被引:2,自引:0,他引:2
采用硫酸铵分级分离,Sephadex G-100凝胶过滤,DEAE-纤维素离子交换层析以及5'-AMP-Sepharose 4B亲和层析,从猪肝微粒体中纯化得到可溶性的NADH-细胞色素b5还原酶,提纯倍数为750-800。总回收率为40%左右。纯化的酶呈典型的黄素蛋白吸收光谱,A273/A460比值为5.8.在SDS-聚丙烯酰胺凝胶电泳板上呈单一的蛋白质区带,分子量为32kd。NADH和2,6- 相似文献
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Svetlana V. Sergeeva Irina A. Slepneva Valery V. Khramtsov 《Free radical research》2013,47(5):491-497
Seleno-organic compounds are known as efficient “scavengers” of peroxynitrite (PN). Here we studied the protective effect of selenolipoic acid (SeLA), the seleno-containing analogue of lipoic acid, on peroxynitrite-dependent inactivation of NADPH-cytochrome P450 reductase. 3-Morpholinosydnonimine hydrochloride (SIN-1) was used as a source of peroxynitrite. The reductase was irreversibly inactivated by PN generated from SIN-1. The inactivation occurred with the rate constant of about 3 × 104M-1s-1. The presence of SeLA at low concentration (0.5 μM) led to synergistic increase of the reductase inactivation by PN. Our results suggest the formation of a reactive derivative of SeLA in the reaction of SeLA with PN, probably selenolseleninate, that mediates the aggravation of reductase inactivation. In the presence of SeLA, the inactivation was reversible under the action of thiols, allowing us to conclude that the observed action of SeLA may be considered as protective. 相似文献
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Svetlana V. Sergeeva Irina A. Slepneva Valery V. Khramtsov 《Free radical research》2001,35(5):491-497
Seleno-organic compounds are known as efficient “scavengers” of peroxynitrite (PN). Here we studied the protective effect of selenolipoic acid (SeLA), the seleno-containing analogue of lipoic acid, on peroxynitrite-dependent inactivation of NADPH-cytochrome P450 reductase. 3-Morpholinosydnonimine hydrochloride (SIN-1) was used as a source of peroxynitrite. The reductase was irreversibly inactivated by PN generated from SIN-1. The inactivation occurred with the rate constant of about 3 × 104M-1s-1. The presence of SeLA at low concentration (0.5 μM) led to synergistic increase of the reductase inactivation by PN. Our results suggest the formation of a reactive derivative of SeLA in the reaction of SeLA with PN, probably selenolseleninate, that mediates the aggravation of reductase inactivation. In the presence of SeLA, the inactivation was reversible under the action of thiols, allowing us to conclude that the observed action of SeLA may be considered as protective. 相似文献
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T Matsubara J Baron L L Peterson J A Peterson 《Archives of biochemistry and biophysics》1976,172(2):463-469
The rate of reduction of cytochrome P450 in hepatic microsomes in the presence of NADPH has been measured with a dual wavelength stopped-flow spectrophotometer. The results obtained, with microsomes prepared from phenobarbital-pretreated rats, indicate that the reduction process is biphasic and most probably composed of two concurrent first-order reactions. The rate constant for the reduction of cytochrome P450 in the fast phase in the presence of ethylmorphine is 1.74 s?1. Since approximately 50% or more of the cytochrome P450 is reduced in the fast phase under these conditions, the rate of reduction of cytochrome P450 is approximately 150 nmol min?1 (mg of protein)?1. Under similar conditions the rate of ethylmorphine N-demethylation is 8.6 nmol min?1 (mg of protein)?1. Thus the rate-limiting step in ethylmorphine N-demethylation cannot be the introduction of the first electron into cytochrome P450 by NADPH-cytochrome P450 reductase. 相似文献
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NADPH-dependen lipid peroxidation catalyzed by purified NADPH-cytochrome C reductase from rat liver microsomes 总被引:5,自引:0,他引:5
A purified preparation of rat liver microsomal NADPH-cytochrome c reductase has been shown to catalyze the NADPH-dependent peroxidation of isolated microsomal lipid. In addition to ADP and ferric ion required for NADPH-dependent lipid peroxidation in whole microsomes, this system requires high ionic strength and a critical concentration of EDTA. The peroxidation activity can be inhibited by superoxide dismutase suggesting that the superoxide anion, produced by this flavoprotein, is involved in the lipid peroxidation reaction. 相似文献
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Several Graves' sera were simultaneously assessed in a bioassay based on the ability of porcine thyroid cells to organify 125I and in a radioreceptor assay for TSH receptor binding activity. Both assay systems were sensitive to 1 mcU/ml (final concentration) of unlabelled bovine TSH. Six Graves' sera were studied in detail over a wide (0-1.0 mcl sera) dose response range in repeat determinations. Two sera exhibited parallel binding and stimulating. However, two sera revealed significant inhibition of 125I-TSH binding prior to the demonstration of stimulation and the other two sera showed stimulatory capabilities before significant binding was evident. IgG was prepared from one serum by ammonium sulphate precipitation and chromatography on Sepharose 6B and then subjected to preparative isoelectric focusing. The isoelectric distribution of the two activities were found to be identical with major peaks of activity at pl=9.5 and pl=8.5. In summary: 1) each Graves' sera exhibits different dose-response curves with respect to binding and stimulation, 2) at certain concentrations of sera, only binding or stimulation were evident, 3) neither assay was consistently more sensitive for the presence of Graves' immunoglobulins, 4) for one Graves' sera, binding and stimulation could not be separated by isoelectric focusing. These studies would suggest each Graves' immunoglobulin has inherently different characteristics in its interaction with the TSH receptor. 相似文献
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Y Nisimoto 《The Journal of biological chemistry》1986,261(30):14232-14239
A covalent complex between purified rat liver microsomal NADPH-cytochrome P-450 reductase and horse cytochrome c was formed through cross-linking studies with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at low ionic strength. The purified cross-linked derivative shows that this product is a 1:1 complex containing one molecule each of the flavoprotein and cytochrome. The covalent complex had almost completely blocked the electron transfer from NADPH to exogenous cytochrome c or the rabbit liver microsomal cytochrome P-450 induced by phenobarbital, indicating that the cross-linked cytochrome c covers the electron-accepting site of the reductase. These results suggest that the covalently cross-linked derivative is a valid model of the noncovalent electron transfer complex. Although the exact number and site of the cross-linked location were not determinable, in cytochrome c the amide bond originates from Lys-13 and in reductase it might be at any one of six different side chain carboxyl groups in the two neighboring cluster acidic residues, Asp-207, -208, and -209, and Glu-213, Glu-214, and Asp-215. It is therefore proposed that the six clustered carboxyl groups on reductase are in an exposed location near the area where one heme edge comes close to the molecular surface. 相似文献
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NADPH-cytochrome c reductase, solubilized by lipase digestion of microsomes prepared from perfused porcine kidney cortex, was purified about 3600-fold to give a turnover number of 1230 nmoles cytochrome c reduced per min per nmole flavin. The kinetic determination of Km and V with respect to NADPH, cytochrome c, and NADH, resulted in values similar to those obtained with purified liver reductase. The kidney microsomal enzyme also exhibited a ping-pong kinetic mechanism for NADPH-mediated cytochrome c reduction.Spectrofluorometric measurements demonstrated the presence of equimolar amounts of FAD and FMN per mole of reductase. The molecular weight was estimated by Sephadex G-200 gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis to be 68,000 and 71,000 g per mole, respectively.Immunochemical techniques, including Ouchterlony double-diffusion studies and inhibition of catalytic activity by antibody to the liver microsomal NADPH-cytochrome c reductase, established the similarity of the purified liver and kidney reductases. 相似文献
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NADPH-cytochrome P450 reductase (CPR) is a membrane-bound flavoprotein that interacts with the membrane via its N-terminal hydrophobic sequence (residues 1-56). CPR is the main electron transfer component of hydroxylation reactions catalyzed by microsomal cytochrome P450s. The membrane-bound hydrophobic domain of NADPH-cytochrome P450 reductase is easily removed during limited proteolysis and is the subject of spontaneous digestion of membrane-binding fragment at the site Lys56-Ile57 by intracellular trypsin-like proteases that makes the flavoprotein very unstable during purification or expression in E. coli. The removal of the N-terminal hydrophobic sequence of NADPH-cytochrome P450 reductase results in loss of the ability of the flavoprotein to interact and transfer electrons to cytochrome P450. In the present work, by replacement of the lysine residue (Lys56) with Gln using site directed mutagenesis, we prepared the full-length flavoprotein mutant Lys56Gln stable to spontaneous proteolysis but possessing spectral and catalytic properties of the wild type flavoprotein. Limited proteolysis with trypsin and protease from Staphylococcus aureus of highly purified and membrane-bound Lys56Gln mutant of the flavoprotein as well as wild type NADPH-cytochrome P450 reductase allowed localization of some amino acids of the linker fragment of NADPH-cytochrome P450 reductase relative to the membrane. During prolong incubation or with increased trypsin ratio, the mutant form showed an alternative limited proteolysis pattern, indicating the partial accessibility of another site. Nevertheless, the membrane-bound mutant form is stable to trypsinolysis. Truncated forms of the flavoprotein (residues 46-676 of the mutant or 57-676 of wild type NADPH-cytochrome P450 reductase) are unable to transfer electrons to cytochrome P450c17 or P4503A4, confirming the importance of the N-terminal sequence for catalysis. Based on the results obtained in the present work, we suggest a scheme of structural topology of the N-terminal hydrophobic sequence of NADPH-cytochrome P450 reductase in the membrane. 相似文献