心磷脂对细胞色素C氧化酶质子泵功能的影响

 用胆酸盐透析法将猪心线粒体细胞色素C氧化酶重组在含心磷脂和二肉豆寇磷脂酰胆碱的脂质体上,以还原态细胞色素C作为酶反应底物,记录脂酶体囊泡外介质液pH的变化,pH下降幅度可以反映细胞色素C氧化酶质子泵的功能。 心磷脂含量不同的细胞色素C氧化酶脂酶体质子泵功能不同。心磷脂含量在10％—40％(w/w)范围内,随心磷脂含量增高,该酶质子泵功能增强;当心磷艏含量超过50％时,该酶质子泵功能却随心磷脂含量的增加表现出下降的趋势。阿霉素可以与心磷脂紧密结合,抑制细胞色素C氧化酶的质子泵功能。然而,少量阿霉素却能增强含70％心磷脂的脂酶体的质子泵功能。     

心磷脂引起细胞色素C的氧化

心磷脂—细胞色素C—细胞色素C氧化酶体系吸收光谱的研究发现:心磷脂与氧化态细胞色素C结合产生230nm吸收峰;心磷脂与还原态细胞色素C作用,230nm吸收值上升,550nm吸收值下降,表明心磷脂可以引起细胞色素C的氧化。     

心磷脂电化学行为的研究

对心磷脂及其与细胞色素C复合物的研究表明,心磷脂具有电化学活性;它与细胞色素C的结合使细胞色素C的克式电位下降、表观电子转移数减少、电极反应变慢。说明心磷脂和细胞色素C之间可能发生了电子转移。     

细胞色素C和含心磷脂的人工脂膜相互作用

 本文报告以芘为荧光探剂,研究细胞色素C和含心磷脂的人工脂膜的相互作用。1.由于芘和细胞色素C的血红素团之间的能量转移,细胞色素C与心磷脂结合引起芘的单体荧光发射峰(395nm)强度下降。这种淬灭效应受脂膜的相行为影响,在液晶相时淬灭效应小于凝胶相;2.氧化态细胞色素C与还原态相比,对心磷脂结合的视和度稍高;3.在以芘的激发二聚体荧光峰(475nm)强度与单体荧光峰强度之比做为脂膜流动性的指标,发现还原态细胞色素C与含心磷脂脂膜结合后引起流动性增加的效应高于氧化态的结合。     

心磷脂-细胞色素C体系CD谱的研究

 线粒体内膜中含有特异的心磷脂是细胞色素C氧化酶活性的必需脂。本工作测定了心磷脂脂质体对细胞色素C溶液园二色(CD)谱的影响,发现心磷脂可引起血色素铁的氧化,并使其轴向配位场强的对称性下降。提示心磷脂可能参与酶和底物之间的电子转移过程。     

谷胱甘肽的抗线粒体脂质过氧化作用

谷胱甘肽是细胞内重要的抗氧化损伤物质之一．以NADH诱导的牛心肌线粒体脂质过氧化体系为模型,研究了谷胱甘肽的抗氧化作用．结果表明,一定浓度的谷胱甘肽能够部分抑制该体系中线粒体的脂质过氧化．保护组的丙二醛含量为损伤组72．5％;线粒体的膨胀度较损伤组降低;细胞色素C氧化酶及ATP酶活力分别较损伤组提高了1．5及2．2倍．     

SO2熏气对油桐叶片细胞膜脂组成和叶绿体超微结构的影响

经SO2熏气处理的油桐叶片电导率增加,叶片中脂质过氧化作用增强,总类脂、极性脂、非极性脂、糖脂含量下降,磷脂含量稍有增加;极性脂和非极性脂中不饱和脂肪酸C18:3含量、C18:3/IUFA、不饱和指标UFA和IUFA值下降,叶绿体结构受到破坏,片层结构模糊不清;随着熏气时间的延长叶绿体中的淀粉颗粒增大,脂滴变小.     

大豆下胚轴线粒体的衰老与膜脂的过氧化作用

离体的大豆下胚轴线粒体,在人工衰老条件下,产生了结构膨胀和细胞色素氧化酶活性的下降。衰老的线粒体也发生膜脂的过氧化作用——丙二醛、脂质的氢过氧化物和荧光脂褐色素明显增加。而且,线粒体衰老时产生的膜脂过氧化产物雨二醛,可能是膜脂的磷脂酰胆碱和磷脂酰乙醇胺中的亚麻酸发生过氧化反应的结果。     

磷脂对琥珀酸-细胞色素c还原酶的作用

琥珀酸细胞色素c还原酶除去90％以上的磷脂后活力丧失约95％。将去脂琥珀酸细胞色素c还原酶与磷脂和辅酶Q_2保温,可恢复其活性。活力恢复程度依赖于磷脂的组成。当磷脂酰胆碱(PC):心磷脂(CL):磷脂酰乙醇胺(PE)=2:2:1时活力恢复最高,比大豆磷脂的效果更为明显,单组分PC,PE或CL恢复活力较差。与酶蛋白紧密结合的CL和PC在活力可逆恢复中有重要作用。     

心磷脂对细胞凋亡作用的研究进展

心磷脂(cardiolipin, CL)是线粒体特异的一类磷脂,它不仅可以维持线粒体的结构,还对电子传递链复合体有影响。在细胞凋亡时,心磷脂会发生一些重分布,比如线粒体内膜外小叶中心磷脂含量的增加及细胞表面心磷脂的出现。同时,凋亡刺激还可以加速心磷脂的代谢循环,使心磷脂在线粒体内的含量降低。在细胞凋亡发生时,心磷脂作为一个信号整合体,对细胞色素c (cytochrome c, CytC)、胱天蛋白酶-8 (caspase-8)和促凋亡蛋白Bid发挥着重要的协调作用。本文主要就心磷脂在细胞凋亡过程中的重要作用及其在癌症中的研究进展进行综述。     

Lipid peroxidation and alterations to oxidative metabolism in mitochondria isolated from rat heart subjected to ischemia and reperfusion.

Ischemia-reperfusion injury to cardiac myocytes involves membrane damage mediated by oxygen free radicals. Lipid peroxidation is considered a major mechanism of oxygen free radical toxicity in reperfused heart. Mitochondrial respiration is an important source of these reactive oxygen species and hence a potential contributor to reperfusion injury. We have examined the effects of ischemia (30 min) and ischemia followed by reperfusion (15 min) of rat hearts, on the kinetic parameters of cytochrome c oxidase, on the respiratory activities and on the phospholipid composition in isolated mitochondria. Mitochondrial content of malonyldialdheyde (MDA), an index of lipid peroxidation, was also measured. Reperfusion was accompanied by a significant increase in MDA production. Mitochondrial preparations from control, ischemic and reperfused rat heart had equivalent Km values for cytochrome c, although the maximal activity of the oxidase was 25 and 51% less in ischemic and reperfused mitochondria than that of controls. These changes in the cytochrome c oxidase activity were associated to parallel changes in state 3 mitochondrial respiration. The cytochrome aa3 content was practically the same in these three types of mitochondria. Alterations were found in the mitochondrial content of the major phospholipid classes, the most pronounced change occurring in the cardiolipin, the level that decreased by 28 and by 50% as function of ischemia and reperfusion, respectively. The lower cytochrome c oxidase activity in mitochondria from reperfused rat hearts could be almost completely restored to the level of control hearts by exogenously added cardiolipin, but not by other phospholipids nor by peroxidized cardiolipin. It is proposed that the reperfusion-induced decline in the mitochondrial cytochrome c oxidase activity can be ascribed, at least in part, to a loss of cardiolipin content, due to peroxidative attack of its unsaturated fatty acids by oxygen free radicals. These findings may provide an explanation for some of the factors that lead to myocardial reperfusion injury.     

The effect of reactive oxygen species generated from the mitochondrial electron transport chain on the cytochrome c oxidase activity and on the cardiolipin content in bovine heart submitochondrial particles

The effect of reactive oxygen species (ROS), produced by the mitochondrial respiratory chain, on the activity of cytochrome c oxidase and on the cardiolipin content in bovine heart submitochondrial particles (SMP) was studied. ROS were produced by treatment of succinate-respiring SMP with antimycin A. This treatment resulted in a large production of superoxide anion, measured by epinephrine method, which was blocked by superoxide dismutase (SOD). Exposure of SMP to mitochondrial mediated ROS generation, led to a marked loss of cytochrome c oxidase activity and to a parallel loss of cardiolipin content. Both these effects were completely abolished by SOD+catalase. Added cardiolipin was able to almost completely restore the ROS-induced loss of cytochrome c oxidase activity. No restoration was obtained with peroxidized cardiolipin. These results demonstrate that mitochondrial mediated ROS generation affects the activity of cytochrome c oxidase via peroxidation of cardiolipin which is needed for the optimal functioning of this enzyme complex. These results may prove useful in probing molecular mechanism of ROS-induced peroxidative damage to mitochondria which have been proposed to contribute to aging, ischemia/reperfusion and chronic degenerative diseases.     

Properties of porcine white adipose tissue and liver mitochondrial subpopulations.

Properties of porcine white adipose tissue heavy and light mitochondrial subpopulations were investigated so as to identify any functional heterogeneity. Liver mitochondrial subpopulations were concurrently evaluated since their properties have been studied in some detail. Mitochondrial subpopulations were isolated by means of differential centrifugation and the relative purity estimated using marker enzymes. Due to the greater contamination of the light mitochondrial fractions, mtDNA content, determined by PCR analysis, was used as a basis to demonstrate any mitochondrial heterogeneity. Enzymatic activity, electron microscopy, lipid analysis and Western blotting were used to characterise the different populations. With the exception of liver cytochrome c oxidase, the enzymatic capacity of adipose and liver heavy mitochondria ranged between approximately two- and threefold higher than the corresponding light fraction. The cardiolipin content and mean mitochondrial diameters paralleled these differences, suggesting an increased mitochondrial mass rather than a functional difference. However, the cytochrome c oxidase activity of the liver heavy mitochondria was 4.75-fold higher relative to the light fraction. A strong correlation between cytochrome c oxidase activity and the subunit I content was evident. Adipose tissue mitochondrial subpopulations would seem to possess a comparable oxidative capacity per gram mitochondrial protein, while liver heavy mitochondria possess an increased oxidative capacity and mass.     

Myocardial ischemia selectively depletes cardiolipin in rabbit heart subsarcolemmal mitochondria

Mitochondria contribute to myocyte injury during ischemia. After 30 and 45 min of ischemia in the isolated perfused rabbit heart, subsarcolemmal mitochondria (SSM), located beneath the plasma membrane, sustain a decrease in oxidative phosphorylation through cytochrome oxidase. In contrast, oxidation through cytochrome oxidase in interfibrillar mitochondria (IFM), located between the myofibrils, remains unaffected. Cytochrome oxidase activity in the intact membrane requires an inner mitochondrial membrane lipid environment enriched in cardiolipin. During ischemia, the content of cardiolipin decreased only in SSM, whereas the content of other phospholipids was preserved. Ischemia did not alter the composition of the cardiolipin that remained in SSM. Cardiolipin content was preserved in IFM during ischemia. Thus cardiolipin is a relatively early target of ischemic mitochondrial damage, leading to loss of oxidative phosphorylation through cytochrome oxidase in SSM.     

Cytochrome oxidase induction after oxidative stress induced by adriamycin in liver of rats fed with dietary olive oil.

The influence of different kinds of dietary fat (8%) and of endogenous lipid peroxidation with regard to cytochrome c oxidase activity and cytochrome a + a3 concentrations in mitochondria from rat liver has been investigated. It was possible to confirm that the dietary fat induced higher phospholipid degradation in mitochondrial membranes; moreover an endogenous oxidative stress induced by adriamycin was able to increase the peroxidative effects. We have found that the peroxidative effects could sometimes induce an apparent enhancement of cytochrome oxidase activity due to a significant increase of cytochrome a + a3 content. This finding lets us suppose that both changes in the lipid environment and some peroxidation damage could occur in the membrane as a consequence of the fat assumed. Furthermore we should suggest that an induction of the synthesis of cytochrome a + a3 might be related to an enhanced production of peroxides at membrane level.     

Lipid composition of mitochondria from bovine heart, liver, and kidney

Highly purified preparations of mitochondria from bovine heart, liver, and kidney were isolated and characterized by electron microscopy, oxidative phosphorylation ability, cytochrome c reductase activity, and cytochrome content. Components of lipid extracts of the preparations were determined by thin-layer chromatography, diethylaminoethyl-cellulose column chromatography, and spectrophotometric procedures. The major phospholipids were identified by their chromatographic behavior, IR spectrometry, and paper chromatography of their hydrolysis products. The lipid content of the mitochondria paralleled that of the components of the electron transfer chain, heart mitochondria being richest and liver mitochondria poorest in lipid. Heart mitochondria contain equal concentrations of coenzyme Q and cholesterol (1%); the highest cholesterol content (4.7%) was found in mitochondria from kidney. The phospholipids of mitochondria from the three organs were qualitatively and quantitatively very similar. The major polar lipid components (cardiolipin, choline glycerophosphatides, and ethanolamine glycerophosphatides) were present in a molar ratio of 1:4:4. It is suggested that mitochondria from different sources contain characteristic lipids, mainly phospholipids, of which cardiolipin is particularly diagnostic of the source of the mitochondria.     

Lipid dependence of mitochondrial monoamine oxidase from rat hepatoma 27 with the use of rat liver lipid exchange proteins]

The ability of liver lipid exchange proteins to introduce foreign phospholipids into intact mitochondria was used for a study of the lipid dependence of monoamine oxidase. Introduction of exogenous phosphatidylcholine into rat hepatoma mitochondria, in which both the monoamine oxidase activity and the phosphatidylcholine content are comparatively low, leads to considerable activation of the enzyme. The introduction of exogeneous phosphatidylserine, phosphatidylethanolamine and cardiolipin has no activating effect. This indicates that the decreased activity of monoamine oxidase in the hepatoma may be due to a low amount of phosphatidylcholine. The method described allows the study in situ of the lipid dependence of non-solubilized membrane-bound enzymes.     

Increase of Lipid Hydroperoxides in Liver Mitochondria and Inhibition of Cytochrome Oxidase by Carbon Tetrachloride -Intoxication in Rats

The cellular localization of lipid hydroperoxides was determined for the first time in mitochondria, microsomes and cytosol of rat liver using a specific method involving chemical derivatization and HPLC. Mitochondria contained the highest level of hydroperoxides. After 6h of intragastric administration of carbon tetrachloride (CCl₄) to rats (2 ml/kg body weight), the concentration of lipid hydroperoxides increased significantly in liver mitochondria and cytochrome oxidase activity was inhibited to 35% of the control rats. The mitochondrial content of haem a decreased to 60% of the control at 12 h of CCl₄ administration. In vitro reaction of mitochondria with CCl₄ caused inactivation of cytochrome oxidase. These observations suggested that cytochrome oxidase and haem a in mitochondria were targets of CCl₄.     

克山病心肌线粒体氧化损伤的实验研究

用克山病区粮喂养豚鼠证明其红细胞、肝等组织及其线粒体、上清中的硒及谷胱甘肽过氧化物酶活性明显降低,心肌线粒体脂质氢过氧化物、荧光色脂等增加、同时膜脂组成异常,内膜界面脂的心磷脂(CL)减少,细胞色素C氧化酶(CCO)活性降低;园二色性(CD)异常,近208nm及222nm区的峰值降低,病区粮加喂青菜的动物,随其线粒体硒及CL水平的增加。CCO活性和CD谱均明显恢复;通过外源性磷脂与纯化CCO的重组复性实验,只有CL可使病区粮组酶活性完全达到正常对照水平。表明心肌线粒体膜结合酶CCO活性的降低是CL含量降低引起酶的二级结构(构象)改变的结果。