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。     

能化态与线粒体及其内膜体膜表面局部脱水

荧光探针ＤＰＥ（Ｄｉｐａｌｍｉｔｏｙｌ－Ｎ－ＤａｎｓｙｌＰｈｏｓｐｈａｔｉｄｙｌｅｔｈａｎｏｌａｍｉｎｅ）标记于鼠肝线粒体及其内膜体膜表面,以测定其膜表面介电常数（ε）的变化与膜能化态的关系．在含有鱼藤酮的线粒体或其内膜体悬液中,加入琥珀酸,使膜处于能化态,可使膜表面。值分别下降１１％和２０％,分别再加ＫＣＮ,ＣＣＣＰ或Ｎｉｇｅｒｉｃｉｎ均能使ε值回升９％;若在上述悬液中,先加ＫＣＮ或ＣＣＣＰ,然后加入琥珀酸,则膜表面ε值无变化。若在线粒体或其内膜体悬液中,先加入ＣＣＣＰ或Ｎｉｇｅｒｉｃｉｎ,两者可使膜表面ε值升高１２％,其后加入鱼藤酮或琥珀酸等,则ε值均无变化,说明呼吸链抑制剂或解偶联剂抑制或解除膜的能化态,均可使膜表面ε值不发生变化。在另一组实验中,先加ＫＣＮ后,再加ＡＴｈ,使线粒体内膜处于能化态,表面ε值下降８％,此时再加寡霉素,则膜表面ε值相反上升１０％。这些实验事实均证明,线粒体膜处于能化态时,ε值下降,解除给化态,使ε值又上升。为能化态与线粒体膜表面水化力下降的相互关系提供了新信息．也为质子泵引起膜表面质子化,进而引起表面脱水,驱动膜融合的理论模型提供了新的证据。     

鱼藤酮对线粒体胆碱-细胞色素c还原酶的抑制

鱼藤酮对新鲜鼠肝线粒体胆碱-细胞色素c还原酶有抑制作用。这种抑制受鱼藤酮与线粒体预保温的影响,然而并不是继发于胆碱的氧化产物三甲铵乙醛的氧化受抑制的结果。另一方面,鱼藤酮与其他呼吸链抑制剂如5乙,5异戊巴比妥酸钠、氰化钾等不同,它有抑制线粒体膨胀的作用。鱼藤酮抑制线粒体胆碱-细胞色素c还原酶的作用很可能与它对线粒体膨胀的抑制有关。     

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。     

力竭性运动对大鼠肝脏线粒体氧化磷酸化偶联的影晌

本文以ＳＤ大鼠三级递增负荷力竭性跑台运动为疲劳模型,分别测定了运动后即刻肝脏线粒体：１．呼吸链复合体Ⅰ＋Ⅲ和Ⅱ＋Ⅲ电子传递与质子泵出比值（Ｈ＋／２ｅ）;２．以苹果酸＋谷氨酸（Ｍ＋Ｇ）和琥珀酸（Ｓ）为底物的呼吸控制：态３呼吸速率（Ｒ３）、态４呼吸速率（Ｒ４）、呼吸控制率（ＲＣＲ）和磷／氧比（Ｐ／Ｏ）。结果表明：两种呼吸底物启动的线粒体态４呼吸速率分别升高６４．４６和２３．５４％（Ｐ＜０．００１和Ｐ＜０．０５）;呼吸链复合体Ⅰ＋Ⅲ和Ⅱ＋Ⅲ的总Ｈ＋／２ｅ分别降低１８．６３和１５．８９％（均Ｐ＜０．０１）。两种呼吸底物的ＲＣＲ和Ｐ／Ｏ呈显著降低（均Ｐ＜０．０５）;Ｍ＋Ｇ为呼吸底物的态３呼吸速率也呈显著增加（Ｐ＜０．０１）,Ｓ为呼吸底物的态３呼吸速率略有增高（Ｐ＞０．０５）。提示,线粒体质子漏增加,呼吸链电子传递与质子泵出偶联程度下降,氧化磷酸化脱偶联导致无效氧耗增多,可能是运动性疲劳状态下线粒体氧利用率下降的重要机制。     

氧化磷酸化作用的研究 Ⅱ.2,4-二硝基苯酚对琥珀酸氧化抑制效应的解除

进一步研究DNP对大白鼠肝脏綫粒体琥珀酸氧化的激活和抑制,发現当琥珀酸氧化已被DNP抑制时琥珀酸脫氫酶并沒有受到明显的抑制。DNP对琥珀酸氧化的抑制可以被α-酮戊二酸、(?)柠檬酸、柠檬酸、丙酮酸、β-羟基丁酸等解除。α-酮戊二酸的解除作用与底物水平磷酸化作用无关,但与脫氫过程有密切关系;当加入0.2mM亚砷酸鈉时,α-酮戊二酸不再能使呼吸恢复。谷氨酸解除DNP对琥珀酸氧化抑制的作用不受天門冬氨酸α-酮戊二酸轉氨酶的抑制剂环絲氨酸的影响,Amytal使呼吸恢复的作用与线粒体內源底物含量有关?疚慕Y果进一步說明琥珀酸氧化需要能量激活,我們认为某些底物脫氫生成的NADH可以通过琥珀酸氧化引起NAD~+需能还原的逆反应生成高能磷酸化合物因而激活了琥珀酸的氧化。通过这样的途径生成高能磷酸化合物可能是对DNP較不敏感的。     

大鼠急性应激时肝线粒体质子跨膜转运活性的调控

大鼠烫伤早期(烫伤后30min),肝线粒体质子和电子传递速度均加快,线粒体能化态跨膜电位降低(均以琥珀酸为底物),线粒体膜脂流动性降低。皮下注射去甲肾上腺素后也有上述现象发生。推测急性应激通过儿茶酚胺类作用于肝细胞,导致线粒体内膜有序性增强所致。     

大鼠急性应激时肝线粒体质子跨膜转动活性的调控

大鼠烫伤早期（烫伤后３０ｍｉｎ）,肝线粒体质子加电子传递速度均加快,线粒体能化态跨膜电位降低（均以琥珀酸为底物）,线粒体膜脂流动性降低。皮下注射去甲肾上腺素后也有上述现象发生。推测急性应激通过儿茶酚胺类作用于肝细胞,导致线粒体内膜有序性增强所致。     

运动性疲劳状态下大鼠心肌线粒体内膜变化的研究

采用递增负荷力竭性运动模型,观察了Ｓｐｒａｇｕｅ－Ｄａｗｌｅｙ大鼠急性运动至力竭后心肌线粒体内膜流动性、ＮＡＤＨ－ＣｏＱ还原酶及ＡＴＰ酶活性的变化。结果表明,大鼠心肌线粒体内膜荧光偏振值较安静时显著增高（Ｐ＜０．０１）,示膜流动性降低。线粒体内膜ＮＡＤＨ－ＣｏＱ还原酶和肌线粒体内膜功能改变,其膜流动性和呼吸链酶活性变化,可能是运动性疲劳的重要膜分子制之一。     

力竭性运动对大鼠肝脏线粒体氧化磷酸化偶联的影响

本文以ＳＤ大鼠三级递增负荷力竭性跑台运动疲劳模型,分别测定了运动后即刻肝脏线粒体;１．呼吸链复合休Ⅰ＋Ⅲ和Ⅱ＋Ⅲ电子传递与质子泵出比值。２．以苹果糖酸＝谷氨酸和琥珀酸为底物的呼吸控制;态３呼吸速度,态４呼吸速率,呼吸控制率和磷／氧比。结果表明;两种呼吸底物启动的线粒体态４呼吸速率分别升高４６．４６和２３．５４％;呼吸链复合体Ⅰ＋Ⅲ和Ⅱ＋Ⅲ的总Ｈ＾３／２ｅ分别降低１８．６３和１５．８９％。     

Malate oxidation, rotenone-resistance, and alternative path activity in plant mitochondria

The effect of cyanide and rotenone on malate (pH 6.8), malate plus glutamate (pH 7.8), citrate, α-ketoglutarate, and succinate oxidation by cauliflower (Brassica oleracea L.) bud, sweet potato (Ipomoea batatis L.) tuber, and spinach (Spinacia oleracea and Kalanchoë daigremontiana leaf mitochondria was investigated. Cyanide inhibited all substrates equally with the exception of malate plus glutamate; in this case, inhibition of O₂ uptake was more severe due to an effect of cyanide on aspartate aminotransferase. Azide and antimycin A gave similar inhibitions with all substrates. Subsequent addition of NAD had no effect with any substrate. Providing that oxalacetate accumulation was prevented, rotenone inhibited all NAD-linked substrates equally and caused ADP:O ratios to decrease by one-third. Addition of succinate to mitochondria oxidizing malate stimulated oxygen uptake, but adding citrate and α-ketoglutarate did not. These results indicate that there is no direct link between malic enzyme and the rotenone- and cyanide-resistant respiratory pathways, and that there is no need to postulate separate compartmentation of malic enzyme and the other NAD-linked enzymes in the matrix.     

Regulation of hydrogen peroxide production by brain mitochondria by calcium and Bax

Abnormal accumulation of Ca2+ and exposure to pro-apoptotic proteins, such as Bax, is believed to stimulate mitochondrial generation of reactive oxygen species (ROS) and contribute to neural cell death during acute ischemic and traumatic brain injury, and in neurodegenerative diseases, e.g. Parkinson's disease. However, the mechanism by which Ca2+ or apoptotic proteins stimulate mitochondrial ROS production is unclear. We used a sensitive fluorescent probe to compare the effects of Ca2+ on H2O2 emission by isolated rat brain mitochondria in the presence of physiological concentrations of ATP and Mg2+ and different respiratory substrates. In the absence of respiratory chain inhibitors, Ca2+ suppressed H2O2 generation and reduced the membrane potential of mitochondria oxidizing succinate, or glutamate plus malate. In the presence of the respiratory chain Complex I inhibitor rotenone, accumulation of Ca2+ stimulated H2O2 production by mitochondria oxidizing succinate, and this stimulation was associated with release of mitochondrial cytochrome c. In the presence of glutamate plus malate, or succinate, cytochrome c release and H2O2 formation were stimulated by human recombinant full-length Bax in the presence of a BH3 cell death domain peptide. These results indicate that in the presence of ATP and Mg2+, Ca2+ accumulation either inhibits or stimulates mitochondrial H2O2 production, depending on the respiratory substrate and the effect of Ca2+ on the mitochondrial membrane potential. Bax plus a BH3 domain peptide stimulate H2O2 production by brain mitochondria due to release of cytochrome c and this stimulation is insensitive to changes in membrane potential.     

Nitric oxide increases oxidative phosphorylation efficiency

We have studied the effect of nitric oxide (NO) and potassium cyanide (KCN) on oxidative phosphorylation efficiency. Concentrations of NO or KCN that decrease resting oxygen consumption by 10–20% increased oxidative phosphorylation efficiency in mitochondria oxidizing succinate or palmitoyl-L-carnitine, but not in mitochondria oxidizing malate plus glutamate. When compared to malate plus glutamate, succinate or palmitoyl-L-carnitine reduced the redox state of cytochrome oxidase. The relationship between membrane potential and oxygen consumption rates was measured at different degrees of ATP synthesis. The use of malate plus glutamate instead of succinate (that changes the H⁺/2e⁻ stoichiometry of the respiratory chain) affected the relationship, whereas a change in membrane permeability did not affect it. NO or KCN also affected the relationship, suggesting that they change the H⁺/2e⁻ stoichiometry of the respiratory chain. We propose that NO may be a natural short-term regulator of mitochondrial physiology that increases oxidative phosphorylation efficiency in a redox-sensitive manner by decreasing the slipping in the proton pumps.     

The relationship between energy generation and cholesterol side-chain cleavage reaction in the mitochondria from human term placenta

1. The interrelationship between progesterone (from cholesterol) biosynthesis and oxidative phosphorylation in human placental mitochondria was examined. 2. ADP and ATP stimulated the malate, succinate and alpha-ketoglutarate-supported progesterone biosynthesis probably via the energy-dependent pyridine nucleotide transhydrogenase activation. The effect of ADP was abolished by rotenone and antimycin in the presence of malate or alpha-ketoglutarate. 3. In the non-energized state of mitochondria malate may supported progesterone biosynthesis by the malic enzyme-dependent pathway. 4. The inhibitory effects of antimycin or cyanide, and the stimulatory effect of rotenone on the succinate-supported progesterone biosynthesis indicate that the succinate to malate conversion is a necessary condition for the stimulation of progesterone biosynthesis from cholesterol. 5. alpha-Ketoglutarate plus malonate did support progesterone biosynthesis also in the presence of ADP or ATP and to a lesser degree in the presence of DNP and rotenone. Arsenate in the presence of alpha-ketoglutarate, malonate, dinitrophenol and rotenone did not affect significantly progesterone biosynthesis. These results indicate that NADPH may be generated also by a non-energy-dependent transhydrogenation in placental mitochondria.     

Putative Structural and Functional Coupling of the Mitochondrial BKCa Channel to the Respiratory Chain

Potassium channels have been found in the inner mitochondrial membranes of various cells. These channels regulate the mitochondrial membrane potential, the matrix volume and respiration. The activation of these channels is cytoprotective. In our study, the single-channel activity of a large-conductance Ca²⁺-regulated potassium channel (mitoBK_Ca channel) was measured by patch-clamping mitoplasts isolated from the human astrocytoma (glioblastoma) U-87 MG cell line. A potassium-selective current was recorded with a mean conductance of 290 pS in symmetrical 150 mM KCl solution. The channel was activated by Ca²⁺ at micromolar concentrations and by the potassium channel opener NS1619. The channel was inhibited by paxilline and iberiotoxin, known inhibitors of BK_Ca channels. Western blot analysis, immuno-gold electron microscopy, high-resolution immunofluorescence assays and polymerase chain reaction demonstrated the presence of the BK_Ca channel β4 subunit in the inner mitochondrial membrane of the human astrocytoma cells. We showed that substrates of the respiratory chain, such as NADH, succinate, and glutamate/malate, decrease the activity of the channel at positive voltages. This effect was abolished by rotenone, antimycin and cyanide, inhibitors of the respiratory chain. The putative interaction of the β4 subunit of mitoBK_Ca with cytochrome c oxidase was demonstrated using blue native electrophoresis. Our findings indicate possible structural and functional coupling of the mitoBK_Ca channel with the mitochondrial respiratory chain in human astrocytoma U-87 MG cells.     

Mechanism of Superoxide Anion Generation in Intact Mitochondria in the Presence of Lucigenin and Cyanide

In the presence of cyanide and various respiratory substrates (succinate or pyruvate + malate) addition of high concentrations of lucigenin (400 microM; Luc2+) to rat liver mitochondria can induce a short-term flash of high amplitude lucigenin-dependent chemiluminescence (LDCL). Under conditions of cytochrome oxidase inhibition by cyanide the lucigenin-induced cyanide-resistant respiration (with succinate as substrate) was not inhibited by uncouplers (FCCP) and oligomycin. Increase in transmembrane potential (Deltaphi) value by stimulating F0F1-ATPase functioning (induced by addition of MgATP to the incubation medium) caused potent stimulation of the rate of cyanide-resistant respiration. At high Deltaphi values (in the presence of MgATP) cyanide resistant respiration of mitochondria in the presence of succinate or malate with pyruvate was insensitive to tenoyltrifluoroacetone (TTFA) or rotenone, respectively. However, in both cases respiration was effectively inhibited by myxothiazol or antimycin A. Mechanisms responsible for induction of LDCL and cyanide resistant mitochondrial respiration differ. In contrast to cyanide-resistant respiration, generation of LDCL signal, that was suppressed only by combined addition of Complex III inhibitors, antimycin A and myxothiazol, is a strictly potential-dependent process. It is observed only under conditions of high Deltaphi value generated by F0F1-ATPase functioning. The data suggest lucigenin-induced intensive generation of superoxide anion in mitochondria. Based on results of inhibitor analysis of cyanide-resistant respiration and LDCL, a two-stage mechanism of autooxidizable lucigenin cation-radical (Luc*+) formation in the respiratory chain is proposed. The first stage involves two-electron Luc2+ reduction by Complexes I and II. The second stage includes one-electron oxidation of reduced lucigenin (Luc(2e)). Reactions of Luc(2e) oxidation involve coenzyme Q-binding sites of Complex III. This results in formation of autooxidizable Luc*+ and superoxide anion generation. A new scheme for lucigenin-dependent electron pathways is proposed. It includes formation of fully reduced form of lucigenin and two-electron-transferring shunts of the respiratory chain. Lucigenin-induced activation of superoxide anion formation in mitochondria is accompanied by increase in ion permeability of the inner mitochondrial membrane.     

Formation of differences in the electric potentials of the membrane vesicle in Staphylococcus aureus

Membrane vesicles isolated from Staphylococcus aureus cells by ultrasonication possess the NADH-, succinate-, and malate oxidase activities, contain cytochromes a and b and have the lipid/protein ratio of 0.12-0.24. Energized membrane vesicles absorb permeant anions of tetraphenylborate and phenyldicarbaundecaborane. This results in the electric field generation with a "plus" sign on the inner side of the membranes. The generation of the membrane potential occurs in response to the addition of a respiratory substrate (NADH, malate, or succinate) and is inhibited by electron transfer inhibitors, such as rotenone, 2-N-nonyl-4-oxyquinoline-N-oxide, cyanide and the protonophore uncoupler, M-chlorinecarbonylcyanidephenylhydrazonium. The generation of the membrane potential takes place during ATP hydrolysis and in the course of the transhydrogenase reaction. The data obtained suggest the similarity of energization systems of St. aureus and those of animal mitochondria.     

Fatty acids decrease mitochondrial generation of reactive oxygen species at the reverse electron transport but increase it at the forward transport

Long-chain nonesterified (“free”) fatty acids (FFA) can affect the mitochondrial generation of reactive oxygen species (ROS) in two ways: (i) by depolarisation of the inner membrane due to the uncoupling effect and (ii) by partly blocking the respiratory chain. In the present work this dual effect was investigated in rat heart and liver mitochondria under conditions of forward and reverse electron transport. Under conditions of the forward electron transport, i.e. with pyruvate plus malate and with succinate (plus rotenone) as respiratory substrates, polyunsaturated fatty acid, arachidonic, and branched-chain saturated fatty acid, phytanic, increased ROS production in parallel with a partial inhibition of the electron transport in the respiratory chain, most likely at the level of complexes I and III. A linear correlation between stimulation of ROS production and inhibition of complex III was found for rat heart mitochondria. This effect on ROS production was further increased in glutathione-depleted mitochondria. Under conditions of the reverse electron transport, i.e. with succinate (without rotenone), unsaturated fatty acids, arachidonic and oleic, straight-chain saturated palmitic acid and branched-chain saturated phytanic acid strongly inhibited ROS production. This inhibition was partly abolished by the blocker of ATP/ADP transfer, carboxyatractyloside, thus indicating that this effect was related to uncoupling (protonophoric) action of fatty acids. It is concluded that in isolated rat heart and liver mitochondria functioning in the forward electron transport mode, unsaturated fatty acids and phytanic acid increase ROS generation by partly inhibiting the electron transport and, most likely, by changing membrane fluidity. Only under conditions of reverse electron transport, fatty acids decrease ROS generation due to their uncoupling action.     

The ROS Production Induced by a Reverse-Electron Flux at Respiratory-Chain Complex 1 is Hampered by Metformin

Mitochondrial reactive oxygen species (ROS) production was investigated in mitochondria extracted from liver of rats treated with or without metformin, a mild inhibitor of respiratory chain complex 1 used in type 2 diabetes. A high rate of ROS production, fully suppressed by rotenone, was evidenced in non-phosphorylating mitochondria in the presence of succinate as a single complex 2 substrate. This ROS production was substantially lowered by metformin pretreatment and by any decrease in membrane potential (Δ < eqid1 > _m), redox potential (NADH/NAD), or phosphate potential, as induced by malonate, 2,4-dinitrophenol, or ATP synthesis, respectively. ROS production in the presence of glutamate–malate plus succinate was lower than in the presence of succinate alone, but higher than in the presence of glutamate–malate. Moreover, while rotenone both increased and decreased ROS production at complex 1 depending on forward (glutamate–malate) or reverse (succinate) electron flux, no ROS overproduction was evidenced in the forward direction with metformin. Therefore, we propose that reverse electron flux through complex 1 is an alternative pathway, which leads to a specific metformin-sensitive ROS production.     

Biochemical properties of rat liver mitochondrial aldehyde dehydrogenase with respect to oxidation of formaldehyde.

The oxidation of formaldehyde by rat liver mitochondria in the presence of 50 mM phosphate was enhanced 2-fold by exogenous NAD+. Absolute requirement of NAD+ for formaldehyde oxidation was demonstrated by depleting the mitochondria of their NAD+ content (4.6 nmol/mg of protein), followed by reincorporation of the NAD+ into the depleted mitochondria. Aldehyde (formaldehyde) dehydrogenase activity was completely abolished in the depleted mitochondria, but the enzyme activity was restored to control levels following reincorporation of the pyridine nucleotide. Phosphate stimulation of formaldehyde oxidation could not be explained fully by the phosphate-induced swelling which enhances membrane permeability to NAD+, since stimulation of the enzyme activity by increased phosphate concentrations was still observed in the absence of exogenous NAD+. The Km for formaldehyde oxidation by the mitochondria was found to be 0.38 nM, a value similar to that obtained with varying concentrations of NAD+; both Vmax values were very similar, giving a value of 70 to 80 nmol/min/mg of protein. The pH optimum for the mitochondrial enzyme was 8.0. Inhibition of the enzyme activity by anaerobiosis was apparently due to the inability of the respiratory chain to oxidize the generated NADH. The inhibition of mitochondrial formaldehyde oxidation by succinate was found to be due to a lowering of the NAD+ level in the mitochondria. Succinate also inhibited acetaldehyde oxidation by the mitochondria. Malonate, a competitive inhibitor of succinic dehydrogenase, blocked the inhibitory effect of succinate. The respiratory chain inhibitors, rotenone, and antimycin A plus succinate, strongly inhibited formaldehyde oxidation by apparently the same mechanism, although the crude enzyme preparation (freed from the membrane) was slightly sensitive to rotenone. The mitochondria were subfractionated, and 85% of the enzyme activity was found in the inner membrane fraction (mitoplast). Furthermore, separation into inner membrane and matrix components indicated a distribution of aldehyde dehydrogenase activity similar to malic dehydrogenase.