高山被孢霉中Δ9脂肪酸脱饱和酶的表达、纯化和其细胞色素b_5功能域的鉴定

在毕赤酵母中表达和纯化源自高山被孢霉ATCC 32222的膜结合Δ9-I脂肪酸脱饱和酶,测定其活性,并探究其细胞色素b_5功能域的性质。构建含有高效纯化标签ZZ-tag的表达载体;用Western blotting和SDS-PAGE筛选Δ9-I脂肪酸脱饱和酶高表达量转化子;通过梯度离心和去垢剂筛选确定膜蛋白质提取条件;采用IgG亲和纯化色谱和阴离子交换色谱对Δ9-I脂肪酸脱饱和酶进行纯化;利用酿酒酵母细胞破碎物为底物考察Δ9-I脂肪酸脱饱和酶活性;通过波长扫描和Na_2S_2O_4还原实验对Δ9-I脂肪酸脱饱和酶细胞色素b_5功能域进行表征。结果显示,目的蛋白质被成功表达并筛选出高表达量转化子;20 000g离心1h为最佳膜分离条件,Fos-Choline-16为最佳去垢剂;纯化后的Δ9-I脂肪酸脱饱和酶结构完整,具有细胞色素b_5功能域;在酿酒酵母提取物中Δ9-I脂肪酸脱饱和酶对C16:0和C18:0底物的转化效率分别为(16.88±9.32)%和(20.61±7.55)%;波长扫描显示Δ9-I脂肪酸脱饱和酶在411nm处有强吸收,并且在Na_2S_2O_4作用下被还原至422nm,说明细胞色素b_5功能域在体外能够被还原。因此,含有细胞色素b_5功能域的脂肪酸脱饱和酶的首次成功表达、纯化和鉴定为亚铁血红素脂肪酸脱饱和酶脱饱和反应机制的研究奠定了基础。     

高山被孢霉亚甲基四氢叶酸脱氢酶的克隆、表达和功能鉴定

叶酸代谢途径中的亚甲基四氢叶酸脱氢酶（MTHFD）可将5,10-亚甲基四氢叶酸氧化为5,10-甲炔基四氢叶酸,此过程会生成NADH或NADPH。对高山被孢霉中的MTHFD基因进行克隆、表达和功能鉴定,可进一步阐明脂质合成所需还原力NADPH的来源。首先对MTHFD序列进行分析,并以pET28a（+）质粒为载体构建了MTHFD的表达载体,然后转化至大肠杆菌BL21中进行诱导表达。进一步利用Ni金属螯合层析纯化目的蛋白,采用比色法分析酶反应产物,表明纯化蛋白质具有MTHFD活性。高山被孢霉MTHFD对NAD⁺和NADP⁺均具有催化能力,但更偏好于将NADP⁺转化为NADPH。最后对高山被孢霉进行发酵培养,发现MTHFD的转录水平在脂质开始积累后发生了明显的上调,表明MTHFD在高山被孢霉脂质合成过程中发挥重要作用,很可能是脂质合成所需NADPH的关键来源。这为对高山被孢霉进行分子改造,使之成为高产各种多不饱和脂肪酸的细胞工程提供了理论依据。     

氯虫苯甲酰胺诱导甜菜夜蛾细胞色素P450基因上调表达

【目的】明确氯虫苯甲酰胺对甜菜夜蛾Spodoptera exigua (Hübner)细胞色素P450基因的诱导表达作用。【方法】采用O-脱乙基香豆素法研究了低剂量氯虫苯甲酰胺处理对甜菜夜蛾幼虫中肠P450s酶活性的影响,应用Real-time PCR方法测定了其对P450基因(CYP9A9, CYP4G37,CYP4S11和CYP6B)和NADPH细胞色素P450还原酶基因(HQ852049)表达的影响。【结果】氯虫苯甲酰胺对甜菜夜蛾P450酶及相关基因的诱导作用均表现出时间效应和剂量效应,。甜菜夜蛾4龄幼虫取食0.02 mg/kg氯虫苯甲酰胺饲料至5龄, 在蜕皮后6-36 h内, 其P450s酶活性增加为对照组的1.90~2.92倍, 诱导效应高于0.01 mg/kg氯虫苯甲酰胺处理组（其P450s酶活性为对照组的1.11~1.62倍）。同时, 0.02 mg/kg氯虫苯甲酰胺处理组甜菜夜蛾中肠P450基因CYP9A9, CYP4G37和CYP6B mRNA的相对表达量分别上升为对照组的1.97~3.95, 2.46~4.29及1.53~4.48倍, NADPH细胞色素P450还原酶基因 HQ852049 的相对表达量亦增加为对照的1.85~4.08倍。【结论】结果提示,氯虫苯甲酰胺可能通过诱导3种P450基因及细胞色素P450还原酶基因 HQ852049 基因mRNA的上调表达而增强了甜菜夜蛾幼虫中肠P450s酶活性。     

过表达亚甲基四氢叶酸脱氢酶对高山被孢霉脂质合成的影响

高山被孢霉是一种富含多不饱和脂肪酸的丝状真菌,但其脂质过程中NADPH的来源还没有研究透彻。以高山被孢霉(尿嘧啶营养缺陷型)作为出发菌株,研究亚甲基四氢叶酸脱氢酶(MTHFD1)对高山被孢霉脂质合成的影响。首先构建了过表达载体pBIG2-ura5s-MTHFD1,采用根癌土壤杆菌介导转化真菌的方法,将二元表达载体转化进高山被孢霉CCFM501中,在筛选培养基SC-CS平板上进行筛选,进而得到稳定遗传MTHFD1基因的过表达菌株(MA-MTHFD1);其次提取MA-MTHFD1菌株基因组进行PCR鉴定,并结合qPCR分析结果,表明MTHFD1基因成功在高山被孢霉中实现了过量表达;最后通过对MA-MTHFD1中的脂肪酸含量、NADPH含量及NADPH合成途径中相关基因转录水平进行分析,研究MTHFD1基因过表达对脂质合成的影响。实验结果表明,过表达MTHFD1基因可以提高高山被孢霉脂质合成能力。与原养型高山被孢霉相比,MA-MTHFD1菌株中脂肪酸含量提高了40.13%,NADPH的含量提高了26.45%,而且NADPH合成途径中其他相关基因苹果酸酶(ME)和异柠檬酸脱氢酶(IDH)的转录水平也发生了上调。这一系列研究结果表明,在高山被孢霉脂质合成还原力形成中,MTHFD1基因起到了关键作用。这为解析高山被孢霉中NADPH来源及深入研究脂质合成机制,从而对其胞内脂肪酸代谢通路进行分子水平上的改建提供了一定的理论依据。     

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

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。     

GhCyt b5基因的克隆及其蛋白参与电子传递功能的分析

从陆地棉品种中棉所35盐胁迫EST文库中筛选到与其它植物高度同源的细胞色素b5蛋白(Cyt b5)基因片段,利用RACE技术,获得Cyt b5基因的cDNA序列,命名为GhCyt b5,基因全长810bp,最大阅读框402 bp,编码由134个氨基酸组成的蛋白质,分子量约为17kDa.将该基因的编码序列插入到原核表达载体pET32a中,构建重组质粒为pET32a-Cyt b5,对不同条件下进行蛋白诱导表达分析,发现在28℃和1 mmol/L IPFG条件下能够获得可溶性的GhCyt b5蛋白.利用Ni2+柱亲和层析纯化和SDS_PAGE鉴定分析表明获得重组蛋白为目的蛋白.通过提取棉花细胞物质为反应介质,进行体外电子传递功能分析,发现GhCyt b5能够从还原态变为氧化态,参与电子的传递功能.     

氧化磷酸化中质子的转运机制介绍和讨论

氧化磷酸化过程中电子传递和磷酸化所伴随的质子（H＋）跨线粒体内膜转运,是生物化学教学中的一个重点和难点。该文介绍参与H＋跨膜（线粒体内膜或细菌质膜）转运的复合体Ⅰ（又称为NADH-Q还原酶或NADH脱氢酶）、复合体Ⅲ（又称为细胞色素还原酶或细胞色素bc1复合体）、复合体Ⅳ（又称为细胞色素氧化酶或细胞色素c氧化酶）和复合体Ⅴ（又称为F1F0-ATP合酶）跨膜转运H＋的机制。     

高山被孢霉Δ6-脂肪酸脱氢酶基因的克隆、结构分析及其功能的研究

从高山被孢霉ATCC1 62 66总DNA中扩增出大小为 1 374bp和 1 947bp的两条特异片段 ,序列分析表明后者在细胞色素b5和组氨酸Ⅰ之间含有一大小为 5 73bp的内含子和两条分别为 1 97bp和 82 8bp的外显子。推导的氨基酸二级结构分析表明 ,该基因有两个长的跨膜疏水区和 3个组氨酸保守区。分别根据D6D内含子及组氨酸Ⅱ区、Ⅲ区的序列设计引物 ,制备不同的探针与高山被孢霉的基因组杂交 ,证明在其基因组中确实存在两个Δ6 脂肪酸脱氢酶基因 ,其中一个基因含有内含子。把不含有内含子的核基因MAGL6 1克隆到的酿酒酵母表达载体pYES2 0中 ,转化到酿酒酵母INVSc1中。对筛选得到的酵母工程菌株进行脂肪酸GC分析 ,检测到了γ 亚麻酸 ,说明克隆的D6D基因MAGL6 1能在酿酒酵母中进行功能性表达。     

转哺乳动物cyp2e1基因烟草植株再生及其分析

哺乳动物肝细胞中cyp2e1基因所编码的蛋白CYP2E1在代谢异型有机物方面起着重要作用,转cyp2e1基因植物可以代谢多种小分子有机污染物;但cyp2e1基因在植物体内的表达调控和代谢机理尚不完全清楚。文中将含有cyp2e1基因的质粒pSLD50-6和对照gus基因的质粒pKH200转入根癌农杆菌GV3101,利用根癌农杆菌转基因技术将cyp2e1基因和对照gus基因成功转入烟草,分别获得了转cyp2e1和gus基因再生植株。选取PCR鉴定的再生植株进行荧光定量PCR(qRT-PCR)分析,结果表明:在转录水平上,转cyp2e1基因烟草中,乙醇处理后cyp2e1基因的表达明显下降,苯和甲苯处理后cyp2e1基因的表达量稍有下降;而丙酮、甲醛处理和缺氧条件下cyp2e1基因的表达有不同程度的升高。此外,苯处理后,转cyp2e1基因烟草中NADPH-P450氧化还原酶和细胞色素b5酶的基因活性显著提高,说明烟草中NADPH-P450氧化还原酶和细胞色素b5酶与CYP2E1酶的解毒过程有关,可能起到哺乳动物体内的NADPH-P450氧化还原酶和细胞色素b5的功能,参与CYP2E1酶催化过程的电子传递链。     

亚致死剂量毒死蜱对飞蝗细胞色素P450酶活性及基因表达的影响

【目的】研究有机磷杀虫剂毒死蜱对飞蝗体内细胞色素P450的影响。【方法】采用酶活力测定法和实时定量PCR技术分别研究了毒死蜱3种亚致死剂量(LD_(10)、LD_(30)和LD_(50))处理飞蝗3龄幼虫24 h后,体内细胞色素P450酶活性及CYP409A1和CYP408B1基因表达量的变化。【结果】不同亚致死剂量毒死蜱处理引起细胞色素P450活性显著性降低,分别为对照组的0.68、0.50和0.62倍。同时通过mRNA水平表达的差异比较显示,飞蝗的两个P450基因CYP409A1和CYP408B1的表达受到抑制,均出现表达量减少的现象。【结论】某些细胞色素P450基因表达受不同亚致死剂量毒死蜱的抑制而使酶的量被降低,从而造成飞蝗整体细胞色素P450酶活性的下降。     

The roles of cytochrome b5 in cytochrome P450 reactions

Cytochrome b(5), a 17-kDa hemeprotein associated primarily with the endoplasmic reticulum of eukaryotic cells, has long been known to augment some cytochrome P450 monooxygenase reactions, but the mechanism of stimulation has remained controversial. Studies in recent years have clarified this issue by delineating three pathways by which cytochrome b(5) augments P450 reactions: direct electron transfer of both required electrons from NADH-cytochrome b(5) reductase to P450, in a pathway separate and independent of NADPH-cytochrome P450 reductase; transfer of the second electron to oxyferrous P450 from either cytochrome b(5) reductase or cytochrome P450 reductase; and allosteric stimulation of P450 without electron transfer. Evidence now indicates that each of these pathways is likely to operate in vivo.     

Chlorzoxazone hydroxylation in microsomes and hepatocytes from cytochrome P450 oxidoreductase‐null mice

Previous studies have demonstrated that the NADH‐dependent cytochrome b₅ electron transfer pathway can support some cytochrome P450 monooxygenases in vitro in the absence of their normal redox partner, NADPH‐cytochrome P450 oxidoreductase. However, the ability of this pathway to support P450 activity in whole cells and in vivo remains unresolved. To address this question, liver microsomes and hepatocytes were prepared from hepatic cytochrome P450 oxidoreductase‐null mice and chlorzoxazone hydroxylation, a reaction catalyzed primarily by cytochrome P450 2E1, was evaluated. As expected, NADPH‐supported chlorzoxazone hydroxylation was absent in liver microsomes from oxidoreductase‐null mice, whereas NADH‐supported activity was about twofold higher than that found in normal (wild‐type) liver microsomes. This greater activity in oxidoreductase‐null microsomes could be attributed to the fourfold higher level of CYP2E1 and 1.4‐fold higher level of cytochrome b₅. Chlorzoxazone hydroxylation in hepatocytes from oxidoreductase‐null mice was about 5% of that in hepatocytes from wild‐type mice and matched the results obtained with wild‐type microsomes, where activity obtained with NADH was about 5% of that obtained when both NADH and NADPH were included in the reaction mixture. These results argue that the cytochrome b₅ electron transfer pathway can support a low but measurable level of CYP2E1 activity under physiological conditions. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:357–363, 2009; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20299     

不同花色矮牵牛细胞色素b5蛋白的cDNA克隆及序列分析

以云南不同花色矮牵牛的花瓣为材料,提取总RNA,用Oligo(dT)作为引物反转录合成cDNA第一链。以此为模板,用根据国外报道的矮牵牛细胞色素b5蛋白的cDNA序列设计合成的引物进行PCR扩,均扩增到一条约450bp的片段,分别克隆到pGEM-T载体上。对重组克隆进行序列分析,结果表明所克隆到的矮牛细胞色素b5蛋白的cDNA的编码区均含有447个核苷酸,编码149个氨基酸残基,与国外报道的一致;但其核苷酸及氨基酸的序列与国外报道的有所不同,即与国外的相比,紫红色、蓝紫色矮牵牛中的该cDNA的核苷酸有1个不同,而氨基酸完全相同;粉红色、白色矮牵牛中的3个核苷酸不同,并导致了2个氨基酸的不同。暗示该基因对花色的调控可能与其编码cDNA的一级结构有关。     

细胞色素b5家族蛋白序列分析

以牛肝微粒体细胞色素b5(CYB5-BOVIN)为切入点,利用生物学信息学方法获得一系列细胞色素b5家族的成员蛋白,同时对蛋白序列进行多重对齐分析及进化分析,借此为细胞色素b5蛋白的分子设计与构建提供指导意义。     

兔肝细胞色素b5在大肠杆菌中的表达

利用体外特定点突变技术,将原核生物核糖体结合位点及十碱基间隔子(spacer)引科兔肝经胞色素b_5 cDNA的5′端,并构建了一个兔红血球b_5cDNA克隆,重组入表达载体pKK223-3质粒中,在大肠杆菌中成功地表达了兔肝和红血球两种形式的b_5蛋白。产量分别为每毫升培养液1.5μg和4.5μg。     

Palmitoyl-CoA Elongation in Brain Microsomes: Dependence on Cytochrome b5 and NADH-Cytochrome b5 Reductase

Experiments were performed to demonstrate the involvement of electron transport system in fatty acid elongation in rat brain microsomes. Mercuric chloride and p-chloromercuriphenylsulfonate, inhibitors on NADH-cytochrome b5 reductase, at 32 microM inhibited NADH-supported palmitoyl-CoA elongation to 30 and 60% of control activity, respectively, whereas NADPH-supported palmitoyl-CoA elongation was unaffected by these mercurials. An antibody to rat liver NADH-cytochrome b5 reductase inhibited brain microsomal NADH-cytochrome b5 reductase activity and NADH-dependent palmitoyl-CoA elongation. Treatment of brain microsomes with trypsin diminished the cytochrome b5 content; NADH- and NADPH-cytochrome c reductase activities were significantly decreased, but the decrease in NADH-cytochrome b5 reductase activity was relatively small. Whereas essentially no incorporation of malonyl-CoA into palmitoyl-CoA was observed with trypsin-treated microsomes, addition of detergent-solubilized cytochrome b5 resulted in a recovery of fatty acid elongation. These results indicate the presence of an electron transport system, NADH-NADH-cytochrome b5 reductase-cytochrome b5-fatty acid elongation, in brain microsomes.     

Human Cytochrome P450 17A1 Conformational Selection: MODULATION BY LIGAND AND CYTOCHROME b5*

Crystallographic studies of different membrane cytochrome P450 enzymes have provided examples of distinct structural conformations, suggesting protein flexibility. It has been speculated that conformational selection is an integral component of substrate recognition and access, but direct evidence of such substate interconversion has thus far remained elusive. In the current study, solution NMR revealed multiple and exchanging backbone conformations for certain structural features of the human steroidogenic cytochrome P450 17A1 (CYP17A1). This bifunctional enzyme is responsible for pregnenolone C17 hydroxylation, followed by a 17,20-lyase reaction to produce dehydroepiandrosterone, the key intermediate in human synthesis of androgen and estrogen sex steroids. The distribution of CYP17A1 conformational states was influenced by temperature, binding of these two substrates, and binding of the soluble domain of cytochrome b₅ (b₅). Notably, titration of b₅ to CYP17A1·pregnenolone induced a set of conformational states closely resembling those of CYP17A1·17α-hydroxypregnenolone without b_5, providing structural evidence consistent with the reported ability of b₅ to selectively enhance 17,20-lyase activity. Solution NMR thus revealed a set of conformations likely to modulate human steroidogenesis by CYP17A1, demonstrating that this approach has the potential to make similar contributions to understanding the functions of other membrane P450 enzymes involved in drug metabolism and disease states.     

Addition of DNA to Cr(VI) and cytochrome b5 containing proteoliposomes leads to generation of DNA strand breaks and Cr(III) complexes

Chromium (Cr) is a cytotoxic metal that can be associated with a variety of types of DNA damage, including Cr-DNA adducts and strand breaks. Prior studies with purified human cytochrome b(5) and NADPH:P450 reductase in reconstituted proteoliposomes (PLs) demonstrated rapid reduction of Cr(VI) (hexavalent chromium, as CrO(4)(2-), and the generation of Cr(V), superoxide (O(2)(*-)), and hydroxyl radical (HO(*)). Studies reported here examined the potential for the species produced by this system to interact with DNA. Strand breaks of purified plasmid DNA increased over time aerobically, but were not observed in the absence of O(2). Cr(V) is formed under both conditions, so the breaks are not mediated directly by Cr(V). The aerobic strand breaks were significantly prevented by catalase and EtOH, but not by the metal chelator diethylenetriaminepentaacetic acid (DTPA), suggesting that they are largely due to HO(*) from Cr-mediated redox cycling. EPR was used to assess the formation of Cr-DNA complexes. Following a 10-min incubation of PLs, CrO(4)(2-), and plasmid DNA, intense EPR signals at g=5.7 and g=5.0 were observed. These signals are attributed to specific Cr(III) complexes with large zero field splitting (ZFS). Without DNA, the signals in the g=5 region were weak. The large ZFS signals were not seen, when Cr(III)Cl(3) was incubated with DNA, suggesting that the Cr(III)-DNA interactions are different when generated by the PLs. After 24 h, a broad signal at g=2 is attributed to Cr(III) complexes with a small ZFS. This g=2 signal was observed without DNA, but it was different from that seen with plasmid. It is concluded that EPR can detect specific Cr(III) complexes that depend on the presence of plasmid DNA and the manner in which the Cr(III) is formed.     

Retrieval from the ER-golgi intermediate compartment is key to the targeting of c-terminally anchored ER-resident proteins

Endoplasmic reticulum (ER) resident proteins may be maintained in the ER by retention, where the leak into post-ER compartments is absent or slow, or retrieval, where a significant leak is countered by retrieval from post-ER compartments. Here the targeting of the C-terminally anchored protein ER-resident protein, cytochrome b5a (cytb5a), considered to be maintained in the ER mainly by the process of retention, is compared with that of sarcolipin (SLN) and phospholamban (PLB); also C-terminally anchored ER-residents. Laser confocal microscopy, and cell fractionation of green fluorescent protein-tagged constructs expressed in COS 7 cells indicate that while calnexin appears to be retained in the ER with no evidence of leak into the ER-Golgi intermediate compartment (ERGIC), significant amounts of cytb5a, SLN, and PLB are detectable in the ERGIC, indicating that there is considerable leak from the ER. This is supported by an in vitro budding assay that shows that while small amounts of calnexin appear in the transport vesicles budding off from the ER, significant amounts of cytb5a and SLN are found in such vesicles. These data support the hypothesis that retrieval plays a major role in ensuring that C-terminally anchored proteins are maintained in the ER.