细胞色素c在氨基酸及二肽修饰金电极上的电化学反应

系统地研究了细胞色素ｃ在多种氨基酸和多肽修饰电极上的电化学反应,并对影响加速细胞色素ｃ电化学反应的因素进行了讨论。     

不同解折叠状态鸡心脱血红素细胞色素c分子的表面性质

脱血红素细胞色素ｃ的构象与其跨膜转运能力是密切相关的。鸡心脱血红素细胞色素ｃ具有较强的自发折叠的能力,在一定条件下可以得到不同解折叠状态的鸡心脱血红素细胞色素ｃ。本文利用疏水层析、与疏水探针１,８—ＡＮＳ的结合以及色氨酸与结合的ＡＮＳ之间的荧光共振能量转移,比较了不同解折叠状态的鸡心脱血红素细胞色素ｃ分子的表面性质。结果表明,部分折叠的鸡心脱血红素细胞色素ｃ分子获得了一种高度动态的结构,形成了动态的疏水核心;同时,它也具有较强的凝集倾向。这些性质与鸡心脱血红素细胞色素ｃ分子较强的自发折叠能力是一致的,为进一步分析鸡心脱血红素细胞色素ｃ分子的构象及其在跨膜转运过程中与脂的相互作用奠定了基础。     

硝基水杨酰胺抑制泛醌-细胞色素c还原酶的作用对氢醌的敏感性

泛醌－细胞色素ｃ还原酶（ＱＣＲ）是线粒体呼吸链的三个能量偶联部位之一,它起着将电子从还原型泛醌传递给细胞色素ｃ（Ｃｙｔ．ｃ）的作用,根据Ｋｉｎｇ和Ｙｕ提出的泛醌结合蛋白理论［１］,泛醌－细胞色素ｃ还原酶中含有泛醌结合蛋白ＱＰｃ．研究表明,泛醌－细胞色...     

脱血红素细胞色素c自发折叠的进一步验证

用胰蛋白酶水解结合聚丙烯酰胺凝胶电泳以及纳秒内源荧光衰减谱分析对鸡心脱血红素细胞色素ｃ在透析复性过程的自发折叠现象作进一步确定。结果显示随着透析复性时间的增加,鸡心脱血红素细胞色素ｃ对胰蛋白酶的水解敏感性逐渐下降,内源荧光寿命值增大,与此对应的马心脱血红素细胞色素ｃ没有发生变化。     

细胞色素C与细胞色素氧化酶之间相互作用的共振RAMAN光谱研究

分别于５１４．５ｎｍ及６０４ｕｍ波长激发下,对游离的细胞色素Ｃ,细胞色素氧化酶以及细胞色素Ｃ和细胞色素氧化酶的复合体的共振拉曼光谱进行了分析比较,在形成复合体时,双方蛋白的共振拉曼谱均有所变化,一个共同的特征性变化是Ａ２ｇｖ２２１１３０ｃｍ－１,ｖ２１１３１２ｃｍ－１,ｖ２０１４００ｃｍ－２,和ｖ１９１５８４ｃｍ－１强度都有增强,其中变化最明显的是Ａ２ｇｖ１９１５８４ｃｍ－１峰,在游离态中,Ｉ１５４０／ｉ１５８２＞１,在结合态中Ｉ１５５０／Ｉ１５８２＜１。     

脱血红素细胞色素c对DMPG膜的插入研究

气－液界面单分子层实验发现鸡心脱血红素细胞色素ｃ对ＤＭＰＧ单分子层表现出很强的插入能力,这种插入能力受亚相溶液离子强度的调节。通过傅里叶变换红外光谱对鸡心脱血红素细胞色素ｃ与ＤＭＰＧ脂质体的作用进行了进一步研究,对磷脂不同区域的红外光谱分析表明鸡心脱血红素细胞色素ｃ不仅与ＤＭＰＧ头部存在静电相互作用而且与其疏水部分也存在相互作用。     

细胞色素c与细胞凋亡

在哺乳动物细胞凋亡的发生过程中,位于线粒体内膜外侧的细胞色素ｃ被释放进入胞质,作为辅助因子参与死亡蛋白酶－３的激活,后者在凋亡过程中起到重要作用。Ｂｃｌ－２、Ｂｃｌ－ＸＬ,Ｂａｘ可阻止或诱导线粒体细胞色素ｃ释放入胞质,这可能是Ｂｃｌ－２,Ｂｃｌ－ＸＬ,Ｂａｘ调节凋亡的机制之一。     

线粒体与细胞凋亡机制

细胞凋亡是生理性的细胞死亡过程,受到多种基因的精确调节,一类被统称为ｃａｓｐａｓｅ的半胱氨酸蛋白酶是细胞凋亡程序的执行者,综们被激活后作用于细胞内的一些蛋白质,经起细胞凋亡。线粒体中含有许多凋亡相关因子,在凋亡信号转导中起着重要作用。细胞受到凋亡刺激后,细胞色素ｃ、ＡＩＦ、ｃａｓｐａｓｅ－９等凋亡相关因子从线粒体中释放出来。细胞色素ｃ通过和Ａｐａｆ－１、ｃａｓｐａｓｅ－９相互作用,激活ｃａｓｐａｓ     

细胞色素c能诱导植物细胞编程性死亡

以悬浮培养的胡萝卜（ＤａｕｃｕｓｃａｒｏｔａＬ．）与烟草（ＮｉｃｏｔｉａｎａｔａｂａｃｕｍＬ．ｃｖ．ＢＹ２）细胞原生质体为材料,加入一定浓度的细胞色素ｃ和ｄＡＴＰ。不同取样时间的ＤＡＰＩ荧光染色与电镜超薄切片观察的结果显示染色质发生凝集、趋边化,最终形成凋亡小体。核酸电泳显示ＤＮＡ发生特异降解并形成电泳“阶梯”（ＤＮＡｌａｄｄｅｒ）。用末端脱氧核糖核酸转移酶介导的ｄＵＴＰ切口末端标记方法（ＴＵＮＥＬ）检测发现ＤＮＡ的３＇ＯＨ断端被原位特异标记。以上结果说明：细胞色素ｃ能诱导植物细胞发生典型的凋亡。     

KATP通道活性对豚鼠结肠带平滑肌细胞电和收缩活动的作用

本文应用平滑肌电活动测量和张力测量技术,利用已知的ＫＡＴＰ通道特异性开放剂（ｃｒｏｍａｋａｌｉｍ）和阻断剂（ｇｌｙｂｅｎｃｌａｍｉｄｅ）,研究ＫＡＴＰ通道对豚鼠结肠带平滑肌细胞电和收缩活动的影响。结果表明：１．ｃｒｏｍａｋａｌｉｍ通过使膜静息电位超极化抑制平滑肌细胞电和收缩活动。２．ｇｌｙｂｅｎｃｌａｍｉｄｅ可以拮抗ｃｒｏｍａｋａｌｉｍ的作用。３．降低灌流液中葡萄糖浓度使平滑肌细胞电和收缩活动下降,ｇｌｙｂｅｎｃｌｍｉｄｅ对此有恢复作用。实验证明：豚鼠结肠带平滑肌细胞膜上存在ＫＡＴＰ通道,ＫＡＴＰ通道通过对膜去极化过程的影响对细胞电和收缩活动起重要作用。     

Bioelectrocatalysis by redox enzymes at modified electrodes

Self-assembled monolayers of thiolated compounds are used as promoters for protein-electrode reactions. They provide an anchor group based on thiol chemisorptions and also a functional group for effective interaction with the protein. These interactions are often governed by electrostatic attraction. For example, for positively charged proteins, such as cytochrome c and the selenoprotein glutathione peroxidase, mercaptoalkanoic acids have been used. Clay modification of the electrode surface has been found to facilitate the heterogeneous electron transfer process for heme proteins, e.g. cytochrome c, cytochrome P450 and myoglobin. Interestingly, nucleic acids at carbon electrodes and thiol-modified double stranded oligonucleotides act as promoters of the redox communication to proteins, whereas the mechanism is still subject to controversy interpretations. By interacting the protein immobilised at the electrode with species in solution, signal chains have been constructed. The interaction can result in a simple co-ordination or redox reaction, depending on the nature of the reaction partners. For analytical purposes, e.g. biosensors, the electrochemical redox conversion of the immobilised protein is evaluated.     

Direct voltammetry of the Chromatium vinosum enzyme, sulfide:cytochrome c oxidoreductase (flavocytochrome c552)

The electrochemistry of the enzyme, sulfide:cytochrome c oxidoreductase, also known as flavocytochrome c552 from the purple sulfur bacterium, Chromatium vinosum, has been studied using several modified electrodes. Direct electron transfer between the heme of the flavocytochrome and an electrode is observed in the presence of a redox-inactive cationic species which promotes the voltammetry of the enzyme. Quasi-reversible electron transfer was achieved using the aminoglycoside, neomycin, as a promoter at either a modified gold or polished edge-plane graphite electrode. Further evidence for direct electron transfer is provided by the catalytic response of the enzyme at the electrode in the presence of substrate. Also reported is the direct spectroelectrochemistry of flavocytochrome c552 at an optically transparent thin layer gold electrode modified with Cys-Glu-Cys in the presence of neomycin.     

Direct electron transfer reactions of cytochrome c553 from Desulfovibrio vulgaris Hildenborough at indium oxide electrodes

The direct, heterogeneous, electron transfer reactions of cytochrome c553 from Desulfovibrio vulgaris Hildenborough have been studied at indium oxide optically transparent electrodes. These reactions have been studied using cyclic voltammetry and derivative cyclic voltabsorptometry and the kinetics of heterogeneous electron transfer is quasi-reversible. The thermodynamics and kinetics of electron transfer by this molecule can be studied at this electrode surface without the need for surface modification or the addition of surface promoters or mediators.     

Electron transfer reactions of metalloproteins at peptide-modified gold electrodes

The electron transfer reactions of four small redox proteins, cytochrome c. ferredoxin, plastocyanin and azurin, have been investigated at novel peptide-modified gold electrodes. These proved to be effective and selective in facilitating electron transfer. Good, quasi-reversible electron transfer was achieved selectively at different peptide-protein configurations by changing the pH or the ionic strength of the solution. The use of peptides as promoters for protein electrochemistry opens up the possibility of designing very specific electrode surfaces for larger molecules like enzymes.     

The Epstein-Barr virus (EBV) BZLF1 immediate-early gene product differentially affects latent versus productive EBV promoters.

The Epstein-Barr virus (EBV) BZLF1 gene product is thought to mediate the disruption of latent EBV infection. We have examined the regulatory effects of BZLF1 by studying its transactivating effects on seven different EBV promoters. We find that whereas the BZLF1 gene product increases the activity of the two early promoters, BMLF1 and BMRF1, it decreases the activity of three latent promoters (the BamHI-C and BamHI-W Epstein-Barr nuclear antigen promoters and the latent membrane protein promoter). The BZLF1-induced changes in promoter-directed chloramphenicol acetyltransferase activity occur in EBV-negative as well as EBV-positive cell lines and are accompanied by a similar change in chloramphenicol acetyltransferase mRNA. Deletion analysis of the BamHI Z fragment indicates that in a portion of the amino-terminal half of the BZLF1 gene product (amino acids 24 to 86) is not essential for positive transactivating effects but is required for down-regulating effects. Thus, different domains of the same EBV immediate-early gene product can either increase the function of EBV promoters active in productive infection or decrease the function of key promoters active in latent infection.     

Deficiency in mRNA splicing in a cytochrome c mutant of neurospora crassa: importance of carboxy terminus for import of apocytochrome c into mitochondria.

Molecular cloning and characterization of cytochrome c cDNA clones of Neurospora crassa wild-type (74A) and a cytochrome c-deficient mutant (cyc1-1) are described. Southern blot analysis of genomic DNA indicates that only one cytochrome c gene exists in the N. crassa genome. The cDNA sequence of the wild-type cytochrome c confirmed the previously determined protein sequence. Sequence analysis of the cyc1-1 cDNA for cytochrome c revealed the presence of a larger open reading frame, owing to the presence of an unspliced intron in the 3' end of the coding region. Splicing of this intron is obviously prevented due to the presence of two base exchanges in the highly conserved intron consensus sequences. Consequently, cyc1-1 synthesizes apocytochrome c with an altered carboxy terminus, 19 amino acids longer than the wild-type cytochrome c, with the final 27 amino acids being of an unrelated sequence. This alteration in the carboxy terminus renders the apocytochrome c incompetent for binding to mitochondria and, consequently, import into mitochondria. Thus, unlike other mitochondrial precursor proteins, where it has been demonstrated that the amino terminus alone is sufficient to target the protein to the mitochondria, an intact carboxy terminus is required for efficient import of apocytochrome c into mitochondria. This is independent confirmation for the view that the import pathway of cytochrome c is unique with respect to all other mitochondrial proteins studied to date.