心磷脂和线粒体内膜

心磷脂是构成线粒体内膜的主要磷脂之一,约75～90%的心磷脂分布在线粒体内膜脂双层的基质面,是线粒体内膜的特征性磷脂。心磷脂使线粒体内膜具有良好的流动性,利于呼吸链各复合物在膜脂双层中的侧向扩散。呼吸链的复合物与心磷脂特异结合才能表现其活性。在一定的条件下,心磷脂亦能形成六角形(?)相,这种多形性特点对离子转运和电子传递有重要意义。     

膜融合的分子机制:线粒体呼吸链不同偶联部位质子泵活性与膜融合程度之间的定量相关性(英文)

我们测定了鼠肝线粒体呼吸链不同偶联部位的质子系活性并通过荧光能量共振转移 法分析了鼠肝线粒体膜与脂质体（二油酰磷脂乙醇胺／心磷脂＝８／２）的膜融合程度。根据测量呼吸链第一段及第二段偶联部位的Ｈ＋／偶联部位的化学计量比值,观察到线粒体呼吸链质子泵的质子（Ｈ＋）泵活性及 Ｈ＋泵出量与膜融合程度呈现线性的定量相关性。这些实验结果进一步支持了我们提出的质子泵诱导膜融合的理论模型（刘树森等,１９８７、１９８９）。     

能化时线粒体内膜脂双分子层结构多相动态

本文报道了鼠肝线粒体内膜体,在琥珀酸氧化或ＡＴＰ水解建立跨膜质子电化学梯度时,膜脂双分子层中ＤＰＨ荧光偏振值出现多个时相动态;ｒ值先迅速下降,再缓慢上升,最终达到高于能化前的ｒ值的恒稳态,表明能化时线粒体内膜脂双分子层有序性在经历短暂下降后,逐渐增大,最终达到更大的结构有序性。在相同时程内呼吸链细胞色素类也经历了相应的多相变化,本文讨论了这两者相关的可能机制,并为呼吸链电子传递机制的动态聚集模型提     

关于呼吸链电子传递与氧化磷酸化机制的教学

近年来,植物生理学中呼吸作用过程及机制研究的新进展和新成果相对较少。在线粒体电子传递系统中,有些传递体的结构和排列、功能尚待进一步确证。目前,对有关呼吸链上伴随着电子传递,线粒体村质中的H~+跨内膜转移到膜间空间、建立质子动力并形成ATP的问题,国内外植物生理学教材有不同提法。在这里,我们试图通过对这些说法的比较分析,以求得到一个比较恰当的说法,这对教学工作可能是有益的。     

线粒体Ca2+转运和通透转变孔道的开放与能量状态

对大鼠肝线粒体Ca2+转运与通透转变孔道(PTP)开放之间的关系, 以及线粒体能量状态对Ca2+转运和PTP开放的影响进行研究. 结果显示, Ca2+诱导的线粒体Ca2+释放(mCICR)能介导PTP开放, 呼吸链电子传递抑制剂抑制mCICR和PTP的开放, 使呼吸链的电子传递局部恢复, mCICR和PTP开放也可部分恢复. mCICR和PTP的开放也能被消除跨膜质子梯差的CCCP抑制, 表明线粒体Ca2+转运和PTP的开放都紧密依赖电子传递和能量偶联.     

线粒体呼吸链复合物I结构和功能的研究进展

线粒体呼吸链复合物I位于线粒体的内膜,是呼吸链中最重要的蛋白复合体之一,可以将电子从NADH传递至CoQ,同时偶联四个质子从线粒体基质泵出至膜间隙,形成跨膜质子梯度,驱动ATP的合成。在目前的研究中,关于复合物I的晶体结构已经比较清楚,包括14个中心亚基,分别构成外周结构域和膜结构域,其中外周结构域负责电子的传递,膜结构域负责质子的泵出。由于在电子传递过程中存在多个中间态阶段,因此复合物I是机体中活性氧产生的主要位点。复合物I也可以通过A/D状态之间的转换,降低活性氧的产生。学者认为复合物I中电子传递产生的静电作用可以改变其结构,从而驱动质子的泵出,但是其具体机制仍不明确。复合物I功能的缺陷是多种神经退行性疾病的诱因,包括阿兹海默症、帕金森等,主要是由于其中不同亚基的点突变导致。本文综述了复合物I结构和功能的研究进展,并对今后的研究做出展望。     

呼吸链底物和抑制剂对线粒体内膜流动性的影响

用DPH和ANS标记大鼠肝线粒体内膜,以稳态荧光偏振法,研究了呼吸链底物和抑制剂对内膜流动性的影响。1.苹果酸+谷氨酸、琥珀酸分别为底物,均能引起内膜流动性增加。2.琥珀酸对含心磷脂的脂质体的膜流动性无影响。3.在鱼藤酮存在的条件下,苹果酸+谷氨酸对内膜流动性的增加作用消失,但琥珀酸的作用仍然存在。有氰化钾时则琥珀酸的作用消失。4.不论外加底物存在与否,鱼藤酮使内膜的流动性下降,而氰化钾则使之增加。抗霉素A亦可使内膜的流动性增加。上述结果表明:线粒体内膜流动性与其功能密切相关。电子沿呼吸链传递使线粒体内膜流动性增加,这种变化可能与呼吸链成分的氧化还原态有关。     

呼吸链电子传递与线粒体内膜流动性

本文报告用稳态荧光各向异性研究呼吸链底物氧化启动电子传递时线粒体内膜的流动性变化。结果表明呼吸链底物使内膜流动性增大,磷脂分子脂酰链的活动度增加(从2位碳到12位碳)。FCCP(p-trifluoromethoxycarbonylcyanide phenylhydrazone)取消H⁺梯度时底物仍可使内膜流动性增加,提示流动性的增加与底物氧化启动的电子沿呼吸链的传递过程密切相关。     

耗竭性运动对大鼠骨骼肌线粒体内膜的影响

观察SD大鼠一次急性运动至力竭后骨骼肌线粒体内膜流动性、NADH-CoQ还原酶及ATP酶活性变化．结果显示,大鼠骨骼肌线粒体内膜微粘度较安静时显著增高,线粒体内膜NADH-CoQ还原酶和ATP酶活性分别较安静时下降34．2％和46．2％．研究提示,耗竭性运动后大鼠骨骼肌线粒体呼吸链内膜分子动力学和呼吸链酶组分活性变化,可能是运动性疲劳重要的膜分子特征．     

线粒体解偶联蛋白UCP2的研究进展

本文综述了线粒体解偶联蛋白2（uncoupling protein2,UCP2）研究方面的进展。UCP2定位于线粒体内膜上,通过消散线粒体内膜的质子梯度调节线粒体的功能,包括线粒体内膜电位、ATP合成、呼吸链ROS产生、线粒体钙库的存储和释放等。目前,UCP2的质子漏机理并不清楚,但体内实验表明UCP2活性可被过氧化物激活。特别是近年来UCP2调控胰岛素分泌方面的研究取得了重要进展。     

质子跨膜转运能够引起艾氏腹水癌细胞质膜表面质子化

荧光探针Ｆｌｕｏｒｅｓｃｅｉｎ－ＰＥ在８ｍｍｏｌ／Ｌ Ｃａ＾２＋促进下,能够标记于艾氏腹水癌细胞质膜表面。在不影响膜结构的条件下,利用微量ＴｒｉｔｏｎＸ－１００消除膜表面与介质之间的质子浓度差,通过酸滴定实验,能够得到膜表面ＰＨ与ＦＰＥ荧光强度之间的相关曲线。根据此曲线测定了艾氏腹水癌细胞质膜表面ＰＨ：当介质ＰＨ为７．２时,其膜表面ＰＨ为６．７,其质子浓度大约为介质中的３倍,质子跨膜转运抑制剂ＣＣ     

Evidence for ΔpH surface component (ΔpH) of proton motive force in ATP synthesis of mitochondria

Background

One of the central debates in membrane bioenergetics is whether proton-dependent energy coupling mechanisms are mediated exclusively by protonic transmembrane electrochemical potentials, as delocalized pmf, ΔµH⁺, or by more localized membrane surface proton pathways, as interfacial pmf, ΔµH^S.

Methods

We measure ?pH^S in rat liver mitoplasts energized by respiration or ATP hydrolysis by inserting pH sensitive fluorescein-phosphatidyl-ethanolamine(F-PE) into mitoplast surface.

Results

In the presence of rotenone and Ap5A, succinate oxidation induces a bi-phasic interfacial protonation on the mitoplast membranes, a fast phase followed by a slow one, and an interfacial pH decrease of 0.5 to 0.9 pH units of mitoplast with no simultaneous pH changes in the bulk. Antimycin A, other inhibitors or uncouplers of mitochondrial respiration prevent the decrease of mitoplast ?pH^S, supporting that ΔµH^S is dependent and controlled by energization of mitoplast membranes. A quantitative assay of ATP synthesis coupled with ?pH^S of mitoplasts oxidizing succinate with malonate titration shows a parallel correlation between ATP synthesis, State 4 respiration and ?pH^S, but not with ?Ψ_E.

General Significance

Our data substantiate ?pH^S as the primary energy source of pmf for mitochondrial ATP synthesis. Evidence and discussion concerning the relative importance and interplay of ?pH^S and ?Ψ_E in mitochondrial bioenergetics are also presented.     

Protein kinase activity and endogenous phosphorylation in subfractions of rat liver mitochondria

Rat liver mitochondria were subfractionated into outer membrane, intermembrane and mitoplast (inner membrane and matrix) fractions. Of the recovered protein kinase activity, 80–90% was found in the intermembrane fraction, while the rest was associated with mitoplast. The intermembrane prostimulated kinase was stimulated by cyclic AMP, while the mitoplast enzyme was stimulated by the nucleotide only after treatment with Triton X-100. Extracted protein kinase resolved into three peaks on DEAE-cellulose chromatography. All three peaks were present both in the intermembrane fraction and in mitoplast. One peak corresponded to the catalytic subunit of cyclic AMP-dependent protein kinase, one was a cyclic AMP-independent enzyme, and the third was the cyclic AMP-dependent type II enzyme. The endogenous incorporation of phosphate was particularly high in the outer mitochondrial membrane, and occurred also in the mitoplast fraction. The incorporation in mitoplasts was to a double band of Mr 47 500, and in outer membranes to apparently heterogeneous material of comparatively low molecular weight.     

Effect of the outer mitochondrial membrane fraction on mitoplast respiration

Additions of the fraction of outer mitochondrial membranes to the mitoplast suspension is shown to bring about an increase of the ADP-stimulated respiration rate, indices of respiration control and uncoupled respiration. This effect is not a result of the cytochrome c presence in the fraction of outer membranes. In the glycerol-containing medium which causes dissociation of intermembrane contacts the coupling effect of outer membranes on mitoplast respiration is not revealed. It is concluded that the outer membrane in contact with the inner one takes part in realization of the mitochondrial coupling.     

Influence of thyroid status on the electrophoretic pattern of rat liver mitochondrial proteins]

Liver mitochondria were isolated from normal and thyroidectomized rats and their protein components analyzed by polyacrylamide gel electrophoresis. In whole mitochondria 35 protein fractions with MW ranging from 10,000 to 135,000 were characterized. In the absence of thyroid hormone secretion, the amount of a MW 54,000 fraction was always decreased. Injection of small doses of 3,5,3'-triiodo-L-thyronine to the thyroidectomized animal restored the quantity of that protein fraction to normal. Isolated outer mitochondrial membranes showed the presence of 20 protein fractions. These fractions revealed no change after thyroidectomy. The mitoplast, which contained 35 fractions, exhibited a decrease of the MW 54,000 component in thyroidectomized rats. The mitoplast was separated into several fractions. Water soluble matrix proteins presented molecular weights ranging between 40,000 and 55,000. Proteins, which were slightly bound to the inner mitochondrial membrane and could be extracted by KCl, presented molecular weights between 25,000 and 45,000. Structural proteins showed a principal specific component of MW equals 23,000. Electrophoretic patterns obtained with these submitochondrial fractions were similar in normal and thyroidectomized animals. The mitoplast fraction which contained the insoluble cytochromes (a, a3, b, c1) was isolated ; its principal constituent, of MW 54,000 was significantly decreased after thyroidectomy. Thus, the lack of thyroid hormone secretion lowered the level of a protein constituent bound to the inner membrane of liver mitochondria. The synthesis of this constituent could be controlled by mitochondrial nucleic acids.     

Protein kinase activity and endogenous phosphorylation in subfractions of rat liver mitochondria

Rat liver mitochondria were subfractionated into outer membrane, intermembrane and mitoplast (inner membrane and matrix) fractions. Of the recovered protein kinase activity, 80-90% was found in the intermembrane fraction, while the rest was associated with mitoplasts. The intermembrane protein kinase was stimulated by cyclic AMP, while the mitoplast enzyme was stimulated by the nucleotide only after treatment with Triton X-100. Extracted protein kinase resolved into three peaks on DEAE-cellulose chromatography. All three peaks were present both in the intermembrane fraction and in mitoplasts. One peak corresponded to the catalytic subunit of cyclic AMP-dependent protein kinases, one was a cyclic AMP-independent enzyme, and the third was the cyclic AMP-dependent type II enzyme. The endogenous incorporation of phosphate was particularly high in the outer mitochondrial membrane, and occurred also in the mitoplast fraction. The incorporation in mitoplasts was to a double band of Mr 47 500, and in outer membranes to apparently heterogeneous material of comparatively low molecular weight.     

Evidence that coupling factor B is bound to the matrix side of the inner mitochondrial membrane

Rotenone-sensitive NADH dehydrogenase activity and Lubrol stimulation of cytochrome oxidase activity were measured to assess the opposite membrane polarity of beef heart mitoplast and inside-out particle preparations. The ATP-Pi exchange activity of mitoplasts was not affected by their incubation at pH 8.9 in the presence of 5 mM EDTA (a treatment known to extract coupling factor B (F beta) from submitochondrial particles), nor was it stimulated by the addition of F beta to intact and alkaline treated mitoplast preparations. In contrast, the exchange activity of inside-out particles was decreased 18 fold by the alkaline/EDTA treatment and was almost completely restored by the addition of F beta to F beta-depleted particles. From these results it is concluded that in beef heart mitochondria, the coupling factor F beta is bound to the matrix-side of the inner mitochondrial membrane.     

The Proteins in Mung Bean Mitochondria

The mung bean mitochondria were obtained by sucrose cushion centrifugation in quite pure form. The outer membrane and the mitoplast were separated by digitonin treatment followed by sucrose density gradient centrifugation. SDS gradient polyacrylamide gel electrophoresis showed that the 42–44 KD protein in mung bean mitochondria was located in mitoplast rather than in the outer membrane. Co-electrophoresis with rabbit muscle actin showed that the rabbit muscle actin coincided with the 44 KD band in plant mitochondria. These results suggest that one of the motive force of the swelling and shrinking of the mitochondria may be generated from the mitochondria themselves.