昆虫细胞色素P450的研究:P450基因的进化

80年代分子生物学方法被用于分离和鉴定特定P450的CDNA或基因组克隆［‘］,至今已知的P450基因包括70个基因家族、127个亚家族的40O多个,基因序列的数量还在迅速增加I’］。不同P450间的进化关系在一些综述中都有涉及［”’,‘］,本文介绍这一主题的一些主要观点,重点放在P450功能的进化及其机制。亚细胞色素P450的基本特征及其分类与命名所有细胞色素P450具有一非共价结合的血红素和环绕高度保守的半跳氨酸的一段26氨基残基的保守序列,这一半跳氨酸提供血红素铁的第5个配体（ligand）。当血红素铁接受电子被还原后再与CO结合产…     

细胞色素P450在生物除污中的应用

人类生存环境中的有毒废物日益增多 ,特别是多环芳烃及多卤代有机物 ,它们的生物利用率低且化学上惰性 ,成为环境污染治理的一大难题 ,迫切需要开发一种有效的途径以解毒或降解这些废物。Ｐ45 0酶系可以催化许多结构不同的物质羟基化、环氧化、脱烷基或脱卤化氢等 ,使其化学性质发生改变。人类活动产生的 2 0 0 0 0 0种环境化合物中大多数是Ｐ45 0的可能底物 ,这使Ｐ45 0在环境的生物除污 (ｂｉｏｒｅｍｅｄｉａｔｉｏｎ)应用中具有广阔前景。1 .天然Ｐ45 0的利用微生物Ｐ45 0无处不在 ,它们具有强的还原和氧化能力 ,对于环境的生物净化十分…     

昆虫细胞色素P450研究:P450基因

细胞色素P450广泛存在于生物界,它因参与许多外来物质和内源性物质的代谢而具有十分重要的作用［1－5］细胞色素P450的研究大约有50多年的历史[3]。60年代的工作主要是对这一血红素蛋白的生物化学和生物物理学特征的了解以及膜结合P450酶系的酶学功能[3]。70年代的研究集中在细胞色素P450酶系的分离纯化及其活性的重组。纯化P450的成功证明了许多P450在物理学和酶学特征方面的不同,也为制备抗体及利用抗体来确定某种P450的存在与数量以及抑制特定P450的酶活性提供了手段,使进一步阐明P450的反应机制成为可能。80年代分子生物学技术的…     

链霉菌细胞色素P450研究进展

链霉菌中存在大量的细胞色素P450,它们在链霉菌次生代谢产物的生物合成和外来化学物质代谢过程中发挥了重要作用.本文综述了链霉菌中发现的细胞色素P450及其功能的研究进展,分析了存在的问题和研究应用前景.     

细胞色素P450的多样性

细胞色素Ｐ４５０的多样性＊邱星辉冷欣夫（中国科学院动物研究所,北京１０００８０）关键词细胞色素Ｐ４５０多样性细胞色素Ｐ４５０是一类以还原态与ＣＯ结合后在波长４５０ｎｍ处有吸收峰的含血红素的单链蛋白质［１～３］。它于１９５８年被发现以来,就引起了人们的...     

植物细胞色素P450

对植物细胞色素P450(CYP450)基因的分离,植物CYP450在苯丙烷类物质、芥子油苷及IAA和萜类等物质的生物合成中的功能,以及对天然生物合成与人工合成物质的解毒功能等研究进展作了简要的综述。指出分离植物细胞色素P450基因,并对其生物学功能进行分析以及植物细胞色素P450降解除草剂的机制及其在环境生物修复等方面的应用是今后一段时间内植物CYP450领域的研究热点。     

细胞色素P450与肿瘤

本文综棕了细胞色素Ｐ４５０同工酶与致癌物代谢、与抗癌药的相互作用以及化的关系,并对调控Ｐ４５０同工酶以防治肿瘤的策略进行了论述。由于Ｐ４５０同工酶具有多态性、工物特异性及可诱导性的特点,在调控Ｐ４５０同工酶以防治肿瘤的问题上,针对不同人群、不同疾病状况及不同用药方案可能需采取抑制或诱导的不同策略。     

工程化P450酶特异性氧化修饰甾体化合物

甾体药物是仅次于抗生素的全球第二大类药物,具有抗炎、抗过敏、调节内分泌等重要功效。对甾体母核特定位点进行有效的氧化修饰,是引入药效活性的关键。研究表明,P450酶是催化甾体特异性氧化的一类关键酶家族。目前,电子传递效率和催化特异性是限制P450酶催化功能的重要因素,会导致目标甾体产物产量低、副产物积累严重等问题。因此,将围绕提高P450酶的催化效率及特异性方面的研究工作,系统综述工程化P450催化甾体类物质的方法策略和相关研究进展,并对催化甾体化合物的P450酶的设计和优化的未来发展进行展望,为该方面工作的深入研究提供指导。     

细胞色素P450调节肝脏药物代谢的途径

大量研究认为细胞色素P450与药物性肝损伤的病理生理过程密切相关,对其在肝损伤的作用已成为当前研究的一个热点.主要介绍细胞色素P450与药物性肝损伤的相关研究进展.     

桔小实蝇CYP4D46的克隆及其组织特异性表达研究

从桔小实蝇Bactrocera dorsalis(Hendel)成虫体内提取总RNA,利用RT-PCR和cDNA末端快速扩增技术获得了一个新的细胞色素P450基因cDNA序列全长.该基因经细胞色素P450基因命名委员会命名为CYP4D46(Gen-Bank登录号:GU292422),其cDNA全长为1717 bp,包含1530 bp的完整开放阅读框(ORF),编码510个氨基酸,理论分子量约为58.40 kD,等电点为8.82.系统发育分析表明该基因与昆虫第4家族P450基因具有较高的同源性.实时定量PCR分析发现,CYP4D46基因在脂肪体中的相对表达量较高,分别是马氏管和中肠内的756倍和60倍,说明CYP4D46可能与脂肪体的重要生理功能相关.     

Expression analysis of the mixed function oxidase system in rat brain by the polymerase chain reaction

Metabolism of therapeutic drugs in the body by the mixed function oxidase system is an important consideration in the analysis of a drug's effectiveness. P450-dependent metabolism within the brain of a neuro-specific drug may affect the drug's course of action. To determine whether cytochrome P450 was expressed in brain, RNA was isolated from the whole brains of rats treated with a variety of known hepatic P450 inducers, including amitriptyline, imipramine, isosafrole, phenobarbital, and -naphthoflavone. The RNA was analyzed for the presence of P450 isozymes by the PCR technique. Differential expression of P450IA1, P450IIB1, P450IIB2, P450IID, and P450IIE1 was detected in the brain samples, depending on the treatment. Cytochrome P450 reductase expression was also detected in the brain samples, giving strong evidence that the brain contains a competent mixed function oxidase system under all conditions studied. (Mol Cell Biochem120: 171–179, 1993)Thesis student of the Graduate School of Biomedical Sciences, the University of Texas Health Science Center at Houston     

An evaluation of the P450 inhibition and induction potential of daptomycin in primary human hepatocytes

Two in vitro studies assessed the potential of daptomycin (Cubicin), a newly marketed antibiotic, to affect the cytochrome P450 (CYP450) isoforms in primary cultured human hepatocytes. Both induction and inhibition of isoforms 1A2, 2A6, 2C9, 2C19, 2D6, 2E1, and 3A4 were evaluated. The highest concentrations of daptomycin used in both the induction and inhibition assays were approximately eight-fold higher than the peak total drug concentration (50-60 microg/mL), or the peak free drug concentration (estimated 5-6 microg/mL), in plasma at the clinical dose regimen of 4 mg/kg qd. Results in primary human hepatocytes indicate that daptomycin, at concentrations up to 400 microg total drug/mL, demonstrated no biologically significant induction of any of the CYP450 isoform activities in comparison with the negative control or known inducers. At daptomycin concentrations up to 40 microg free drug/mL, no biologically significant inhibition of the activities of these CYP450 isoforms was observed as compared with known inhibitors. The human hepatocyte results demonstrate that daptomycin has no effects on hepatic CYP450-mediated drug metabolism and, therefore, suggest that daptomycin is unlikely to show potential for pharmacokinetic interactions with concomitantly administered drugs that are metabolized by CYP450 isoforms.     

Preparation of Cell Wall Fractions by Various Ways and Activity of Bound Saccharase in Sugar Beet Seedlings

Washed cells of facultative methylotrophs which have the serine pathway showed high activities for l-methionine formation from dl-homocysteine, in the presence of methanol as methyl donor. Strain FM 518, isolated from soil and identified as a bacterium belonging to the genus Pseudomonas, showed the highest activity for l-methionine formation and was used as the parental strain for breeding the l-methionine-producing mutants. An ethionine-resistant mutant, FE 244, derived from strain FM 518, accumulated 0.8 mg/ml l-methionine in a methanol-medium under optimum conditions.     

Evidence for the involvement of a distinct form of cytochrome p450 3a in the oxidation of digitoxin by rat liver microsomes

The preceding paper (B. Gemzik, D. Greenway, C. Nevins, and A. Parkinson (1992). Regulation of two electrophoretically distinct proteins recognized by antibody against rat liver cytochrome P450 3A1. J. Biochem. Toxicol, 7 (43–52).) described the regulation of two rat liver microsomal proteins (50- and 51-kDa) recognized by antibody against P450 3A1. It was also shown that changes in the levels of the 51-kDa 3A protein were usually paralleled by changes in the rate of testosterone 2β-, 6β-, and 15β-hydroxylation. The present study demonstrates that age- and sex-dependent changes in the 50-kDa protein were paralleled by changes in the rate of digitoxin oxidation to digitoxigenin bisdigitoxoside. Induction or suppression of the 50-kDa protein by treatment of rats with various xenobiotics were also paralleled by changes in the rate of digitoxin oxidation. These results suggest that, contrary to previous assumptions, the conversion of digitoxin to digitoxigenin bisdigitoxoside and the conversion of testosterone to 2β-, 6β- and 15β-hydroxytestosterone are primarily catalyzed by different forms of P450 3A. Further evidence for this coclusion was obtained from studies in which the suicide inhibitor, chloramphenicol, was administered to mature female rats previously treated with pregnenolone-16α-carbonitrile (PCN), which induces both the 50-kDa and the 51-kDa protein. Treatment of mature female rats with PCN alone caused a marked increase (16- to 18-fold) in the 6β-hydroxylation of testosterone and the rate of digitoxin oxidation. Treatment of PCN-induced rats with chloramphenicol caused a ～70% decrease in liver microsomal testosterone 6β-hydroxylation, but had no effect on the rate of conversion of digitoxin to digitoxigenin bisdigitoxoside. The oxidation of testosterone by purified 3A1 (a 51-kDa protein) was also inhibited by chloramphenicol in a time- and reduced nicotinamite adenine dinucleotide phosphate (NADPH)-dependent manner. In addition to testosterone and chloramphenicol, purified 3A1 also metabolized trole-andomycin, but it was unable to convert digitoxin to digitoxigenin bisdigitoxoside. Testosterone inhibited the microsomal oxidation of digitoxin, but digitoxin did not inhibit testosterone oxidation. This suggests that testosterone is a substrate for the 3A enzyme that metabolizes digitoxin, but that this form of P450 3A does not contribute significantly to testosterone oxidation by rat liver microsomes. We propose that the 2SbT-, 6β-, and 15β-hydroxylation of testosterone by rat liver microsomes is primarily catalyzed by the 51-kDa 3A proteins (either 3A1 or 3A2 depending on the source of microsomes), whereas digitoxin oxidation is primarily catalyzed by the 50-kDa protein.     

High-throughput cytochrome P450 (CYP) inhibition screening via cassette probe-dosing strategy: II. Validation of a direct injection/on-line guard cartridge extraction–tandem mass spectrometry method for CYP2D6 inhibition assessment

A highly efficient direct injection/on-line guard cartridge extraction–tandem mass spectrometry (DI/GCE–MS–MS) method has been validated for high-throughput evaluation of cytochrome P450 (CYP) 2D6 inhibition potential using human hepatic microsomes and 96-well microtiter plates. Microsomal incubations were terminated with formic acid, centrifuged, and the resulting supernatants were injected for DI/GCE–MS–MS analysis. Due to the novel use of an extremely short C₁₈ guard cartridge, this method exhibits several advantages, such as no sample preparation, excellent on-line extraction, short run time (2.5 min), and minimized source contamination and performance deterioration. The DI/GCE–MS–MS method demonstrates acceptable accuracy and precision for the quantification of dextrorphan, a marker metabolite of dextromethorphan mediated by CYP2D6, in microsomal incubations. The CYP2D6 inhibition assay has been validated using quinidine as a known selective inhibitor of the isoform. The IC₅₀ value (0.20 μM) measured by the new method is in good agreement with the literature value (0.22 μM).     

Sunflower cDNA Encoding New Cytochrome P450

The primary structure of the cDNA clone SF28 was determined in sunflower (Helianthus annuusL.) flowers. The clone comprises a 874-bp insert corresponding to 227 amino acid residues of the C-terminal part of the cytochrome P450 gene. The sunflower cytochrome P450 was considerably different from the already known plant and animal cytochromes P450.     

Preliminary crystallographic analysis of an enzyme involved in erythromycin biosynthesis: Cytochrome P450eryF

Cytochrome P450eryF was overexpressed in Escherichia coli and purified in high yield. Crystals of the protein in the presence of the substrate, 6-deoxyerythronolide B, have been obtained by the hanging drop vapor diffusion method, using polyethylene glycol 4000 as a precipitant. The crystals belong to the orthorhombic space group P2₁2₁2₁ with unit cell dimensions of a = 54.16 Å, b = 79.67 Å, and c = 99.48 Å and one molecule per asymmetric unit. A complete native data set has been collected to a resolution of 2.1 Å, and anomalous dispersion difference Patterson maps have revealed the location of the single heme iron atom. © 1994 Wiley-Liss, Inc.     

Productive and non-productive complexes in cytochrome P450-containing systems

The equilibrium dissociation constants K_D, the complex association / dissociation rate constants (k _on /k _off) and lifetimes of the complexes of redox partners were measured for three cytochrome P450-containing monooxygenase systems (P450cam, P450scc, and P450 2B4) under hydroxylation conditions. The Q parameter representing the ratio of protein-protein complex lifetime (τ _lT ) to time required for a single hydroxylation cycle (τ_turnover) was introduced for estimation of productivity of complexes formed within the systems studied. The Q parameter was insignificantly changed upon transition from the oxidation to hydroxylation conditions. Lifetimes (τ _lT ) for the binary complexes formed within the P450cam and the P450scc systems obligatory requiring an intermediate electron transfer protein between the reductase and cytochrome P450 could not realize hydroxylation reactions for substrates with known τ_turnover and so they were non-productive while the binary complexes formed within the P450 2B4 system, not requiring such intermediate electron-transfer protein, appeared to be productive. Formation of ternary complexes was demonstrated under hydroxylation conditions in all three systems. Analysis of Q values led to the conclusion that the ternary complexes formed within the P450cam and the P450scc systems were productive. In the case of the P450 2B4 system, more than half (about 60%) ternary complexes were also found to be productive.