猪心线粒体Fo的纯化、重建及其质子转运功能

比较了猪心线粒体F_oF₁-ATPase膜部分F_o的四种纯化方法.结果表明,用NaBr从亚线粒体除去F_oF₁-ATPase的水溶性部分F₁-ATPase后,再以CHAPS增溶,并经蔗糖梯度离心,可获得高纯度的F_o.SDS-聚丙烯酰胺凝胶电泳鉴定表明,纯化的F_o含有b、OSCP（寡霉素敏感授予蛋白）、d、a、e、F₆、IF₁、A6L和c等9种亚基.用去污剂稀释法将纯化的F_o在脂质体上重建后,重建F_o表现较高的被动转运质子活性.这为在体外深入研究F_o的活性、构象与膜脂的关系,以及F_o与F₁-ATPase的组装等提供了很好的实验模型.     

酵母线粒体的磷脂转运

线粒体是一种由两层膜包被的细胞器,其功能和结构的稳定性取决于线粒体膜上精确的磷脂组成及分布。线粒体膜上的大部分脂类物质由内质网合成,既而转运到线粒体。而部分脂类利用内质网上产生的前体,在线粒体内膜上合成。由此可见,线粒体膜脂的生物合成需要线粒体与内质网以及线粒体外膜(outer mitochondrial membrane, OMM)与内膜(inner mitochondrial membrane, IMM)之间进行大量的脂质转运。目前认为,这种运输过程既可在拴系因子(tether factors)形成的膜结合部位(membrane contact sites, MCSs)内发生,也可借助脂质转运蛋白(lipid transfer proteins, LTPs)完成。近年来,研究者以酵母为对象,建立了多种线粒体磷脂转运(phospholipid trafficking)的模型,这使人们初步理解了线粒体磷脂转运的机制。本综述总结了酵母线粒体磷脂转运的最新发现,并对这些磷脂转运的模型进行了讨论,以期为今后深入了解线粒体脂类代谢提供参考。     

线粒体转运蛋白质家族

线粒体转运蛋白质家族（mitochondrial transporter family）等可溶性物质载体（solute carrier,SLC）,主要包括SLC25,广泛存在于真核生物线粒体中,负责可溶性物质跨线粒体内膜的转运。SLC25家族成员拥有相似的结构特征、种类繁多的转运底物,并与细胞的多种生理功能密切相关。有研究表明,SLC25家族蛋白质的缺失或突变可导致多种代谢性疾病或神经系统疾病的发生。     

神经元轴突线粒体长距离转运的力生物学

作为极性细胞,神经元由胞体、网状的树突和细长并具有分支的轴突构成。完成分化的神经元在脊椎动物的整个生命周期中要保持正常的生理功能,需要大量的能量来维持静息电位与突触传递。神经元主要依赖于线粒体氧化磷酸化产生的ATP提供能量。神经元通过长距离运输与锚定将健康的线粒体运送并富集到轴突分支突触前末梢等能量消耗较大的区域,同时将轴突末梢老化或受损的线粒体反向转运到胞体进行清除。本文结合作者自己的研究从力学的观点,论述在驱动力作用下线粒体是如何克服阻力在轴突进行长距离运输。综述内容包括微管的极性、微管马达蛋白、线粒体衔接蛋白复合物、线粒体与锚定蛋白相互作用、细胞内阻力、线粒体与内质网的相互作用、线粒体生成、裂变、融合、分裂、质量控制等方面。这些新颖的观点将为认识由线粒体运输障碍引起的神经系统疾病提供重要的参考。     

大鼠心肌线粒体L-Arg/ NO系统对线粒体Ca2+转运功能的影响

目的和方法采用大鼠心肌线粒体体外孵育的方法,观察线粒体L-精氨酸/一氧化氮系统对线粒体Ca2+转运功能的影响.结果NO生成的底物L-Arg (10-4 mol/L)、外源性NO供体硝普纳(5×10-7 mol/L)孵育的线粒体NO-2的生成量分别高于对照组66%、89% (P<0.01);钙含量较对照组分别低40%、54% (P<0.01); 线粒体Ca2+的摄入量较对照组分别减少67%、85%(P<0.01), 线粒体Ca2+释放率(11%、8%)降低与对照组(14%)相比差异显著(P<0.05、P<0.01).NO合酶抑制剂左旋硝基精氨酸甲酯(L-NAME, 10-4 mol/L)与相同浓度的L-Arg共同孵育的线粒体,明显抑制了L-Arg对线粒体的效应,与单纯L-Arg组比较,NO2生成减少,线粒体钙含量和反映线粒体45 Ca2+的摄入与释放能力都接近对照组水平.结论心肌线粒体L-精氨酸/一氧化氮系统参与了线粒体对心肌细胞Ca2+浓度的调节,其生理和病理生理意义值得进一步探讨.     

内毒素休克大鼠肝线粒体质子跨膜转运的改变

用稳态荧光探针标记技术动态观察内毒素休克大鼠肝亚线粒体质子跨膜转运的变化.发现,休克5 h ATP、NADH和琥珀酸钠所致的9-氨基-6-氯-2-甲氧基吖啶(ACMA）最大荧光淬灭值（ΔA_max）显著低于对照组(P＜0.05)、最大荧光淬灭时间（T_ΔAmax）、半数荧光淬灭时间（T_1/2ΔAmax）非常显著延长(P＜0.01),肝线粒体质子跨膜转运能力下降;膜脂分子烃链和膜脂深层次流动性下降;线粒体膜PLA₂活性增加;血浆脂质过氧化产物MDA和线粒体MDA含量均显著增加.可能膜脂质过氧化和磷脂酶A₂的水解是引起内毒素休克肝线粒体质子转运功能改变的重要因素.     

细胞间线粒体转运的研究进展

线粒体是真核生物能量代谢的重要细胞器,是细胞进行氧化磷酸化生成ATP的主要场所.他参与完成细胞能量代谢、维持离子浓度梯度、传递细胞凋亡信号等生理功能.阿尔茨海默病、帕金森病、心肌梗塞等疾病与线粒体功能异常相关.近年来发现,由创伤或炎症造成脑、心脏、肺缺氧时在细胞间会发生线粒体转移.线粒体转移,作为一种进化上保守的现象可能与神经降解、心血管疾病等相关.     

Escherichia coli YidC is a membrane insertase for Sec-independent proteins

YidC is a recently discovered bacterial membrane protein that is related to the mitochondrial Oxa1p and the Alb3 protein of chloroplasts. These proteins are required in the membrane integration process of newly synthesized proteins that do not require the classical Sec machinery. Here we demonstrate that YidC is sufficient for the membrane integration of a Sec-independent protein. Microgram amounts of the purified single-spanning Pf3 coat protein were efficiently inserted into proteoliposomes containing the purified YidC. A mutant Pf3 coat protein with an extended hydrophobic region was inserted independently of YidC into the membrane both in vivo and in vitro, but its insertion was accelerated by YidC. These results show that YidC can function separately from the Sec translocase to integrate membrane proteins into the lipid bilayer.     

The distribution of phospholipids on the mitochondrial inner membrane from the brains of goldfish acclimated at 5 and 30°C

1. 1.|The instantaneous addition of phospholipase C to goldfish brain inner mitochondrial vesicles resulted in a partial loss of ferricyanide reduction. The retained activity rose when the phospholipase concentration and incubation period were increased.

2. 2.|No impairment of ferricyanide reduction was observed when Antimycin A-treated inner membrane vesicles were incubated with the phospholipase at 0.05 and 0.2 U mg⁻¹ to 20 min. However, higher concentrations of the hydrolytic enzyme caused a considerable elevation in reduction and is attributed to a deterioration in vesicular integrity.

3. 3.|The hydrolysis of phospholipids from the inner membrane revealed that the phospholipase, at concentrations above 0.2 U mg⁻¹ (minimum of 10 min incubation), resulted in removal of over 50% lipid phosphate. It was suggested that the phospholipase, at such levels, is accessible to the interior of the vesicles.

4. 4.|With the exception of cardiolipin and phosphatidyl inositol, the phospholipase was observed to be non-specific to the phospholipids from mitochondrial inner membrane treated with Triton X-100.

5. 5.|The phospholipids are asymmetrically arranged across the inner membrane and their distribution is dependent on acclimation temperature.

6. 6.|It is suggested that the compositional redistribution of the phospholipids, when acclimation temperature was changed, is necessary to stabilize the bilayer conformation of the membrane in concurrence with maintaining fluidity and functional capacity.

Direct measurements of ferric reductase activity of human 101F6 and its enhancement upon reconstitution into phospholipid bilayer nanodisc

We studied human 101F6 protein to clarify its physiological function as a ferric reductase and its relationship to tumor suppression activity. We found for the first time that purified 101F6 both in detergent micelle state and in phospholipid bilayer nanodisc state has an authentic ferric reductase activity by single turnover kinetic analyses. The kinetic analysis on the ferrous heme oxidation of reduced 101F6 upon the addition of a ferric substrate, ferric ammonium citrate (FAC), showed concentration-dependent accelerations of its reaction with reasonable values of K_M and V_max. We further verified the authenticity of the ferric reductase activity of 101F6 using nitroso-PSAP as a Fe²⁺-specific colorimetric chelator. 101F6 in nanodisc state showed higher efficiency for FAC than in detergent micelle state.     

基于Blast-GO的蛋白质亚线粒体定位预测

本文建立了一个最新的蛋白质亚线粒体定位数据集,包含4个亚线粒体定位的1 293条序列,结合基因本体(GO)信息和同源信息对线粒体蛋白质进行特征提取,利用支持向量机算法建立分类器,经Jackknife检验,对于4个亚线粒体位置的总体预测准确率为93.27%,其中3个亚线粒体位置的总体预测准确率为94.73%.     

Mg~(2+)对线粒体H~+-ATP酶的F_O在脂质体重建时的影响

线粒体ATP合成酶是由具有H~+转运活性的F_0亚基,可溶性的催化中心F_1和连接二者的致寡霉素敏感蛋白(OSCP)所组成. 将纯化的猪心线粒体H—ATP酶复合体的F_0亚基,用胆酸盐透析法在有Mg~(2+)和无Mg~(2+)条件下在大豆磷脂脂质体上重建得脂酶体(L·F_0).用探剂9-AA荧光淬灭法和电权法测定了两种脂酶体的质子转运活力.由两种方法所得的实验结果均表明,在透返介质中加入1mmolmg~(2+)条件下形成的脂酶体(L·F_0)+Mg~(2+)较无Mg~(2+)者的质子转运活性明显增加.前者的荧光强度变化较后者增加约30%;由电极法测得的质子转运的初速度,前者为5nmolH~+′sF_0,后者为3nmolH~+′s·nmolF_O,质子转运活性高约一倍.这进一步支持Mg~(2+)通过调节脂的物理状态而诱导F_O具有较适合的构象,并进而将这一影响传递至F_1,使整个H~+—AhP酶具有较高活性的假设.