Measurement of ATP production and respiratory chain enzyme activities in mitochondria isolated from small muscle biopsy samples

A set of methods suitable for assessment of respiratory chain function in mitochondria isolated from 25mg of muscle is described. This set of methods includes determination of the mitochondrial ATP production rate (MAPR) and the activities of the respiratory chain complexes I, I+III, II+III, and IV and citrate synthase. MAPR is determined with an optimized version of a luminometric method previously described. The optimized method measures 50-220% higher activities than the original method. The highest MAPRs are recorded using the substrate combinations glutamate+succinate and N,N,N(1),N(1)-tetramethyl-1,4-phenyldiamine+ascorbate. The respiratory chain complex activities are determined with standard spectrophotometric methods, adapted to an automated photometer. The sensitivity in the determination of complex I, I+III, and II+III activities was increased considerably by pretreating the samples with saponin. The set of methods was evaluated on double biopsy samples from five healthy volunteers and showed coefficients of variation between 7 and 14% when citrate synthase was used as reference base. All of the various measures of mitochondrial function showed high correlation coefficients to each other (r=0.84-0.98; p<0.01). It is concluded that the set of methods is suitable for diagnosis of mitochondrial disorders in adults and small children.     

Respiratory chain supercomplexes in plant mitochondria

Supercomplexes are defined associations of protein complexes, which are important for several cellular functions. This "quintenary" organization level of protein structure recently was also described for the respiratory chain of plant mitochondria. Except succinate dehydrogenase (complex II), all complexes of the oxidative phosphorylation (OXPOS) system (complexes I, III, IV and V) were found to form part of supercomplexes. Compositions of these supramolecular structures were systematically investigated using digitonin solubilizations of mitochondrial fractions and two-dimensional Blue-native (BN) polyacrylamide gel electrophoresis. The most abundant supercomplex of plant mitochondria includes complexes I and III at a 1:2 ratio (I1 + III2 supercomplex). Furthermore, some supercomplexes of lower abundance could be described, which have I2 + III4, V2, III2 + IV(1-2), and I1 + III2 + IV(1-4) compositions. Supercomplexes consisting of complexes I plus III plus IV were proposed to be called "respirasome", because they autonomously can carry out respiration in the presence of ubiquinone and cytochrome c. Plant specific alternative oxidoreductases of the respiratory chain were not associated with supercomplexes under all experimental conditions tested. However, formation of supercomplexes possibly indirectly regulates alternative respiratory pathways in plant mitochondria on the basis of electron channeling. In this review, procedures to characterize the supermolecular organization of the plant respiratory chain and results concerning supercomplex structure and function are summarized and discussed.     

线粒体呼吸链膜蛋白复合体的结构

线粒体作为真核细胞的重要“能量工厂”,是细胞进行呼吸作用的场所,呼吸作用包括柠檬酸循环和氧化磷酸化两个过程,其中氧化磷酸化过程的电子传递链（又称线粒体呼吸链）位于线粒体内膜上,由四个相对分子质量很大的跨膜蛋白复合体（Ⅰ、Ⅱ、Ⅲ、和Ⅳ）、介于Ⅰ／Ⅱ与Ⅲ之间的泛醌以及介于Ⅲ与Ⅳ之间的细胞色素c共同组成。线粒体呼吸链的功能是进行生物氧化,并与称之为复合物V的ATP合成酶（磷酸化过程）相偶联,共同完成氧化磷酸化过程,并生产能量分子ATP。线粒体呼吸链的结构生物学研究对于彻底了解电子传递和能量转化的机理是至关重要的,本文分别论述线粒体呼吸链复合体Ⅰ、Ⅱ、Ⅲ和Ⅳ的结构,并跟踪线粒体呼吸链超复合体的结构研究进展。     

NMDA and non-NMDA receptor-mediated differential Ca2+ load and greater vulnerability of motor neurons in spinal cord cultures

Glutamate receptor activated neuronal cell death has been implicated in the pathogenesis of motor neuron disease but the molecular mechanism responsible for neuronal dysfunction needs to be elucidated. In the present study, we examined the contribution of NMDA and non-NMDA sub-types of glutamate receptors in selective vulnerability of motor neurons. Glutamate receptor activated Ca2+ signaling, mitochondrial functions and neurotoxicity in motor neurons and other spinal neurons were studied in mixed spinal cord primary cultures. Exposure of cells to glutamate receptor agonists glutamate, NMDA and AMPA elevated the intracellular Ca2+, mitochondrial Ca2+ and caused mitochondrial depolarization and cytotoxicity in both motor neurons and other spinal neurons but a striking difference was observed in the magnitude and temporal patterns of the [Ca2+]i responses between the two neuronal cell types. The motor neurons elicited higher Ca2+ load than the other spinal neurons and the [Ca2+]i levels were elevated for a longer duration in motor neurons. AMPA receptor stimulation was more effective than NMDA. Both the NMDA and non-NMDA receptor antagonists APV and NBQX inhibited the Ca2+ entry and decreased the cell death significantly; however, NBQX was more potent than APV. Our results demonstrate that both NMDA and non-NMDA sub-types of glutamate receptors contribute to glutamate-mediated motor neuron damage but AMPA receptors play the major role. AMPA receptor-mediated excessive Ca2+ load and differential handling/regulation of Ca2+ buffering by mitochondria in motor neurons could be central in their selective vulnerability to excitotoxicity.     

Copper deprivation potentiates oxidative stress in HL-60 cell mitochondria.

Cytochrome-c oxidase is the copper-dependent terminal respiratory complex (complex IV) of the mitochondrial electron transport chain whose activity in a variety of tissues is lowered by copper deficiency. Because inhibition of respiratory complexes increases the production of reactive oxygen species by mitochondria, it is possible that copper deficiency increases oxidative stress in mitochondria as a consequence of suppressed cytochrome-c oxidase activity. In this study, the activities of respiratory complex I + III, assayed as NADH:cytochrome-c reductase, complex II + III, assayed as succinate:cytochrome-c reductase, complex IV, assayed as cytochrome-c oxidase, and fumarase were measured in mitochondria from HL-60 cells that were grown for seven passages in serum-free medium that was either unsupplemented or supplemented with 50 n M CuSO4. Fumarase activity was not affected by copper supplementation, but the complex I + III:fumarase and complex IV:fumarase ratios were reduced 30% and 50%, respectively, in mitochondria from cells grown in the absence of supplemental copper. This indicates that copper deprivation suppressed the electron transfer activity of copper-independent complex I + III as well as copper-dependent complex IV. Manganese superoxide dismutase (MnSOD) content was also increased 49% overall in the cells grown in the absence of supplemental copper. Furthermore, protein carbonyl groups, indicative of oxidative modification, were present in 100-kDa and 90-kDa proteins of mitochondria from copper-deprived cells. These findings indicate that in cells grown under conditions of copper deprivation that suppress cytochrome-c oxidase activity, oxidative stress in mitochondria is increased sufficiently to induce MnSOD, potentiate protein oxidation, and possibly cause the oxidative inactivation of complex I.     

Wy-14,643 and fenofibrate inhibit mitochondrial respiration in isolated rat cardiac mitochondria

We investigated the direct effects of two selective PPARalpha ligands, fenofibrate and Wy-14,643, on mitochondrial respiratory function using isolated rat cardiac mitochondria. Isolated left ventricular mitochondria were incubated with increasing concentrations of fenofibrate or Wy-14,643 (10, 100, and 500 microM) and mitochondrial respiration determined using: malate/glutamate (complex I), succinate (complex II) and palmitoyl-l-carnitine as oxidative substrates. Our data show that acute exposure to Wy-14,643 and fenofibrate differentially perturb cardiac mitochondrial respiration i.e., fenofibrate more potently inhibited mitochondrial respiration and bioenergetic capacity compared to Wy-14,643. Moreover, we found that both agents increased uncoupling of mitochondrial oxidative phosphorylation.     

The mitochondrial respiratory chain is partially organized in a supercomplex assembly: kinetic evidence using flux control analysis

The model of the respiratory chain in which the enzyme complexes are independently embedded in the lipid bilayer of the inner mitochondrial membrane and connected by randomly diffusing coenzyme Q and cytochrome c is mostly favored. However, multicomplex units can be isolated from mammalian mitochondria, suggesting a model based on direct electron channeling between complexes. Kinetic testing using metabolic flux control analysis can discriminate between the two models: the former model implies that each enzyme may be rate-controlling to a different extent, whereas in the latter, the whole metabolic pathway would behave as a single supercomplex and inhibition of any one of its components would elicit the same flux control. In particular, in the absence of other components of the oxidative phosphorylation apparatus (i.e. ATP synthase, membrane potential, carriers), the existence of a supercomplex would elicit a flux control coefficient near unity for each respiratory complex, and the sum of all coefficients would be well above unity. Using bovine heart mitochondria and submitochondrial particles devoid of substrate permeability barriers, we investigated the flux control coefficients of the complexes involved in aerobic NADH oxidation (I, III, IV) and in succinate oxidation (II, III, IV). Both Complexes I and III were found to be highly rate-controlling over NADH oxidation, a strong kinetic evidence suggesting the existence of functionally relevant association between the two complexes, whereas Complex IV appears randomly distributed. Moreover, we show that Complex II is fully rate-limiting for succinate oxidation, clearly indicating the absence of substrate channeling toward Complexes III and IV.     

Malonaldehyde acts as a mitochondrial toxin: Inhibitory effects on respiratory function and enzyme activities in isolated rat liver mitochondria

Malonaldehyde (MDA) is a product of oxidative damage to lipids, amino acids and DNA, and accumulates with aging and diseases. MDA can possibly react with amines to modify proteins to inactivity enzymes and also modify nucleosides to cause mutagenicity. Mitochondrial dysfunction is a major contributor to aging and age-associated diseases. We hypothesize that accumulated MDA due to mitochondrial dysfunction during aging targets mitochondrial enzymes to cause further mitochondrial dysfunction and contribute to aging and age-associated diseases. We investigated the effects of MDA on mitochondrial respiration and enzymes (membrane complexes I, II, III and IV, and dehydrogenases, including alpha-ketoglutaric dehydrogenase (KGDH), pyruvate dehydrogenase (PDH), malate dehydrogenase (MDH)) in isolated rat liver mitochondria. MDA showed a dose-dependent inhibition on mitochondrial NADH-linked respiratory control ratio (RCR) and ADP/O ratio declined from the concentrations of 0.2 and 0.8 micromol/mg protein, respectively, and succinate-linked mitochondrial RCR and ADP/O ratio declined from 1.6 and 0.8 micromol/mg protein. MDA also showed dose-dependent inhibition on the activity of PDH, KGDH and MDH significantly from 0.1, 0.2 and 2 micromol/mg protein, respectively. Activity of the complexes I and II was depressed by MDA at 0.8 and 1.6 micromol/mg protein. However, MDA did not affect activity of complexes III and IV in the concentration range studied (0-6.4 micromol/mg protein). These results suggest that MDA can cause mitochondrial dysfunction by inhibiting mitochondrial respiration and enzyme activity, and the sensitivity of the enzymes examined to MDA is in the order of PDH>KGDH>complexes I and II>MDH>complexes III and IV.     

Chromium(VI) interaction with plant and animal mitochondrial bioenergetics: a comparative study

The mechanism of Cr(VI)-induced toxicity in plants and animals has been assessed for mitochondrial bioenergetics and membrane damage in turnip root and rat liver mitochondria. By using succinate as the respiratory substrate, ADP/O and respiratory control ratio (RCR) were depressed as a function of Cr(VI) concentration. State 3 and uncoupled respiration were also depressed by Cr(VI). Rat mitochondria revealed a higher sensitivity to Cr(VI), as compared to turnip mitochondria. Rat mitochondrial state 4 respiration rate triplicated in contrast to negligible stimulation of turnip state 4 respiration. Chromium(VI) inhibited the activity of the NADH-ubiquinone oxidoreductase (complex I) from rat liver mitochondria and succinate-dehydrogenases (complex II) from plant and animal mitochondria. In rat liver mitochondria, complex I was more sensitive to Cr(VI) than complex II. The activity of cytochrome c oxidase (complex IV) was not sensitive to Cr(VI). Unique for plant mitochondria, exogenous NADH uncoupled respiration was unaffected by Cr(VI), indicating that the NADH dehydrogenase of the outer leaflet of the plant inner membrane, in addition to complexes III and IV, were insensitive to Cr(VI). The ATPase activity (complex V) was stimulated in rat liver mitochondria, but inhibited in turnip root mitochondria. In both, turnip and rat mitochondria, Cr(VI) depressed mitochondrial succinate-dependent transmembrane potential (Deltapsi) and phosphorylation efficiency, but it neither affected mitochondrial membrane permeabilization to protons (H+) nor induced membrane lipid peroxidation. However, Cr(VI) induced mitochondrial membrane permeabilization to K+, an effect that was more pronounced in turnip root than in rat liver mitochondria. In conclusion, Cr(VI)-induced perturbations of mitochondrial bioenergetics compromises energy-dependent biochemical processes and, therefore, may contribute to the basal mechanism underlying its toxic effects in plant and animal cells.     

Effect of Ischemic Preconditioning on Mitochondrial Dysfunction and Mitochondrial P53 Translocation after Transient Global Cerebral Ischemia in Rats

Transient global brain ischemia induces dysfunctions of mitochondria including disturbance in mitochondrial protein synthesis and inhibition of respiratory chain complexes. Due to capacity of mitochondria to release apoptogenic proteins, ischemia-induced mitochondrial dysfunction is considered to be a key event coupling cerebral blood flow arrest to neuronal cell death. Ischemic preconditioning (IPC) represents an important phenomenon of adaptation of central nervous system (CNS) to sub-lethal short-term ischemia, which results in increased tolerance of CNS to the lethal ischemia. In this study we have determined the effect of ischemic preconditioning on ischemia/reperfusion-associated inhibition of mitochondrial protein synthesis and activity of mitochondrial respiratory chain complexes I and IV in the hippocampus of rats. Global brain ischemia was induced by 4-vessel occlusion in duration of 15 min. Rats were preconditioned by 5 min of sub-lethal ischemia and 2 days later, 15 min of lethal ischemia was induced. Our results showed that IPC affects ischemia-induced dysfunction of hippocampal mitochondria in two different ways. Repression of mitochondrial translation induced during reperfusion of the ischemic brain is significantly attenuated by IPC. Slight protective effect of IPC was documented for complex IV, but not for complex I. Despite this, protective effect of IPC on ischemia/reperfusion-associated changes in integrity of mitochondrial membrane and membrane proteins were observed. Since IPC exhibited also inhibitory effect on translocation of p53 to mitochondria, our results indicate that IPC affects downstream processes connecting mitochondrial dysfunction to neuronal cell death.     

"Respirasome"-like supercomplexes in green leaf mitochondria of spinach

Higher plant mitochondria have many unique features compared with their animal and fungal counterparts. This is to a large extent related to the close functional interdependence of mitochondria and chloroplasts, in which the two ATP-generating processes of oxidative phosphorylation and photosynthesis, respectively, take place. We show that digitonin treatment of mitochondria contaminated with chloroplasts from spinach (Spinacia oleracea) green leaves at two different buffer conditions, performed to solubilize oxidative phosphorylation supercomplexes, selectively extracts the mitochondrial membrane protein complexes and only low amounts of stroma thylakoid membrane proteins. By analysis of digitonin extracts from partially purified mitochondria of green leaves from spinach using blue and colorless native electrophoresis, we demonstrate for the first time that in green plant tissue a substantial proportion of the respiratory complex IV is assembled with complexes I and III into "respirasome"-like supercomplexes, previously observed in mammalian, fungal, and non-green plant mitochondria only. Thus, fundamental features of the supramolecular organization of the standard respiratory complexes I, III, and IV as a respirasome are conserved in all higher eukaryotes. Because the plant respiratory chain is highly branched possessing additional alternative enzymes, the functional implications of the occurrence of respiratory supercomplexes in plant mitochondria are discussed.     

Physical and functional interaction of NCX1 and EAAC1 transporters leading to glutamate-enhanced ATP production in brain mitochondria

Glutamate is emerging as a major factor stimulating energy production in CNS. Brain mitochondria can utilize this neurotransmitter as respiratory substrate and specific transporters are required to mediate the glutamate entry into the mitochondrial matrix. Glutamate transporters of the Excitatory Amino Acid Transporters (EAATs) family have been previously well characterized on the cell surface of neuronal and glial cells, representing the primary players for glutamate uptake in mammalian brain. Here, by using western blot, confocal microscopy and immunoelectron microscopy, we report for the first time that the Excitatory Amino Acid Carrier 1 (EAAC1), an EAATs member, is expressed in neuronal and glial mitochondria where it participates in glutamate-stimulated ATP production, evaluated by a luciferase-luciferin system. Mitochondrial metabolic response is counteracted when different EAATs pharmacological blockers or selective EAAC1 antisense oligonucleotides were used. Since EAATs are Na(+)-dependent proteins, this raised the possibility that other transporters regulating ion gradients across mitochondrial membrane were required for glutamate response. We describe colocalization, mutual activity dependency, physical interaction between EAAC1 and the sodium/calcium exchanger 1 (NCX1) both in neuronal and glial mitochondria, and that NCX1 is an essential modulator of this glutamate transporter. Only NCX1 activity is crucial for such glutamate-stimulated ATP synthesis, as demonstrated by pharmacological blockade and selective knock-down with antisense oligonucleotides. The EAAC1/NCX1-dependent mitochondrial response to glutamate may be a general and alternative mechanism whereby this neurotransmitter sustains ATP production, since we have documented such metabolic response also in mitochondria isolated from heart. The data reported here disclose a new physiological role for mitochondrial NCX1 as the key player in glutamate-induced energy production.     

Astrocyte-derived nitric oxide causes both reversible and irreversible damage to the neuronal mitochondrial respiratory chain

Cytokine-stimulated astrocytes produce nitric oxide (NO), which, along with its metabolite peroxynitrite (ONOO(-)), can inhibit components of the mitochondrial respiratory chain. We used astrocytes as a source of NO/ONOO(-) and monitored the effects on neurons in coculture. We previously demonstrated that astrocytic NO/ONOO(-) causes significant damage to the activities of complexes II/III and IV of neighbouring neurons after a 24-h coculture. Under these conditions, no neuronal death was observed. Using polytetrafluoroethane filters, which are permeable to gases such as NO but impermeable to NO derivatives, we have now demonstrated that astrocyte-derived NO is responsible for the damage observed in our coculture system. Expanding on these observations, we have now shown that 24 h after removal of NO-producing astrocytes, neurons exhibit complete recovery of complex II/III and IV activities. Furthermore, extending the period of exposure of neurons to NO-producing astrocytes does not cause further damage to the neuronal mitochondrial respiratory chain. However, whereas the activity of complex II/III recovers with time, the damage to complex IV caused by a 48-h coculture with NO-producing astrocytes is irreversible. Therefore, it appears that neurons can recover from short-term damage to mitochondrial complex II/III and IV, whereas exposure to astrocytic-derived NO for longer periods causes permanent damage to neuronal complex IV.     

Changes of Respiratory Chain Activity in Mitochondrial and Synaptosomal Fractions Isolated from the Gerbil Brain After Graded Ischaemia

Abstract: In this study we have examined (1) the integrated function of the mitochondrial respiratory chain by polarographic measurements and (2) the activities of the respiratory chain complexes I, II–III, and IV as well as the ATP synthase (complex V) in free mitochondria and synaptosomes isolated from gerbil brain, after a 30-min period of graded cerebral ischaemia. These data have been correlated with cerebral blood flow (CBF) values as measured by the hydrogen clearance technique. Integrated functioning of the mitochondrial respiratory chain, using both NAD-linked and FAD-linked substrates, was initially affected at CBF values of ∼35 ml 100 g⁻¹ min⁻¹, and declined further as the CBF was reduced. The individual mitochondrial respiratory chain complexes, however, showed differences in sensitivity to graded cerebral ischaemia. Complex I activities decreased sharply at blood flows below ∼30 ml 100 g⁻¹ min⁻¹ (mitochondria and synaptosomes) and complex II–III activities decreased at blood flows below 20 ml 100 g⁻¹ min⁻¹ (mitochondria) and 35–30 ml 100 g⁻¹ min⁻¹ (synaptosomes). Activities declined further as CBF was reduced below these levels. Complex V activity was significantly affected only when the blood flow was reduced below 15–10 ml 100 g⁻¹ min⁻¹ (mitochondria and synaptosomes). In contrast, complex IV activity was unaffected by graded cerebral ischaemia, even at very low CBF levels.     

Effect of sydnone SYD-1, a mesoionic compound, on energy-linked functions of rat liver mitochondria

An important antitumour effect of SYD-1 (3-[4-chloro-3-nitrophenyl]-1,2,3-oxadiazolium-5-olate) has been shown. We now report the effects of this mesoionic compound on mitochondrial metabolism. SYD-1 (1.5 micromol mg(-1) protein) dose-dependently inhibited the respiratory rate by 65% and 40% in state 3 using sodium glutamate and succinate, respectively, as substrates. Phosphorylation efficiency was depressed by SYD-1, as evidenced by stimulation of the state 4 respiratory rate, which was more accentuated with glutamate ( approximately 180%) than with succinate ( approximately 40%), with 1.5 micromol mg(-1) protein of SYD-1. As a consequence of the effects on states 3 and 4, the RCC and ADP/O ratios were lowered by SYD-1 using both substrates, although this effect was stronger with glutamate. The formation of membrane electrical potential was inhibited by approximately 50% (1.5 micromol SYD-1mg(-1) protein). SYD-1 interfered with the permeability of the inner mitochondrial membrane, as demonstrated by assays of mitochondrial swelling in the presence of sodium acetate and valinomycin +K(+). SYD-1 (1.5 micromol mg(-1) protein) inhibited glutamate completely and succinate energized-mitochondrial swelling by 80% in preparations containing sodium acetate. The swelling of de-energized mitochondria induced by K(+) and valinomycin was inhibited by 20% at all concentrations of SYD-1. An analysis of the segments of the respiratory chain suggested that the SYD-1 inhibition site goes beyond the complex I and includes complexes III and IV. Glutamate dehydrogenase was inhibited by 20% with SYD-1 (1.5 micromol mg(-1) protein). The hydrolytic activity of complex F(1)F(o) ATPase in intact mitochondria was greatly increased ( approximately 450%) in the presence of SYD-1. Our results show that SYD-1 depresses the efficiency of electron transport and oxidative phosphorylation, suggesting that these effects may be involved in its antitumoural effect.     

Identification of respiratory complexes I and III as mitochondrial sites of damage following exposure to ionizing radiation and nitric oxide.

In 32D cl 3 hematopoietic progenitor cells, the overexpression of manganese superoxide dismutase (MnSOD, SOD2), the enzyme normally found in mitochondria, protects against the damaging effects of ionizing radiation. In the presence of a nitric oxide donor, which exacerbates the damage, inhibition of mitochondrial function can be demonstrated to be associated with respiratory complexes I (NADH dehydrogenase) and III (cytochrome c reductase), but not II (succinate dehydrogenase), IV (cytochrome c oxidase), or V (ATP synthase). The same pattern of inhibition is observed in the case of isolated bovine heart mitochondria exposed to ionizing radiation and the nitric oxide donor. The addition of authentic peroxynitrite (ONO2(-)) to isolated mitochondria also results in damage to complexes I and III (but not II, IV, and V), as shown by assays of electron-transfer activities and electron paramagnetic resonance (EPR) spectroscopic measurements, suggesting ONO2(-) to be responsible for most of the observed radiation damage in both the cultured cell lines and isolated mitochondria. It is argued that, in general, production of ONO2(-) is an important contributor to radiation damage in biological systems and the implications of these findings in relation to possible mechanisms of oxidant-linked apoptosis are briefly considered.     

Complex I Controls Mitochondrial and Plasma Membrane Potentials in Nerve Terminals

Reductions in the activities of mitochondrial electron transport chain (ETC) enzymes have been implicated in the pathogenesis of numerous chronic neurodegenerative disorders. Maintenance of the mitochondrial membrane potential (Δψ_m) is a primary function of these enzyme complexes, and is essential for ATP production and neuronal survival. We examined the effects of inhibition of mitochondrial ETC complexes I, II/III, III and IV activities by titrations of respective inhibitors on Δψ_m in synaptosomal mitochondria. Small perturbations in the activity of complex I, brought about by low concentrations of rotenone (1–50 nM), caused depolarisation of Δψ_m. Small decreases in complex I activity caused an immediate and partial Δψ_m depolarisation, whereas inhibition of complex II/III activity by more than 70% with antimycin A was required to affect Δψ_m. A similarly high threshold of inhibition was found when complex III was inhibited with myxothiazol, and inhibition of complex IV by more than 90% with KCN was required. The plasma membrane potential (Δψ_p) had a complex I inhibition threshold of 40% whereas complex III and IV had to be inhibited by more than 90% before changes in Δψ_p were registered. These data indicate that in synaptosomes, both Δψ_m and Δψ_p are more susceptible to reductions in complex I activity than reductions in the other ETC complexes. These findings may be of relevance to the mechanism of neuronal cell death in Parkinson’s disease in particular, where such reductions in complex I activity are present.

     

线粒体电子传递系统的组装及其生物学意义

电子传递链亦称呼吸链,由位于线粒体内膜的I、II、III、IV 4种复合物组成,负责电子传递和产生质子梯度。电子主要从复合物I进入电子传递链,经复合物III传递至复合物IV。电子传递系统的组装是一个十分复杂的过程,目前已知主要有约69个结构亚基以及至少16个组装因子参与了人类复合物I、III、IV的组装,这些蛋白质由核基因组与线粒体基因组共同编码。对线粒体电子传递系统的蛋白质组成及其结构已研究得较为清楚,但对它们的组装了解得还比较初步。许多人类线粒体疾病是由于电子传递系统的功能障碍引起的,其中又有许多是由于该系统中一个或多个部件的错误组装引起的。研究这些缺陷不仅能够加深对线粒体疾病发病机理的了解,也有助于揭示线粒体功能的调控机制。将着重对电子传递系统复合物的组装及其与人类疾病关系的研究进展进行综述。     

An Evaluation of the Measurement of the Activities of Complexes I-IV in the Respiratory Chain of Human Skeletal Muscle Mitochondria

The measurement of individual respiratory chain complexes is an important component of the investigation of diseases due to mitochondrial dysfunction. We have evaluated assays which measure complexes I to IV in human skeletal muscle mitochondria and in addition optimized these assays to provide sensitive and reliable diagnostic techniques, particularly in situations where a partial interruption at a single complex needs to identified. Using several established methods of membrane disruption we have found that optimal activities of complexes I and II are obtained by freeze-thawing the mitochondria in hypotonic potassium phosphate buffer, whereas complex III and IV activities are markedly increased by the addition of the detergent n-dodecyl-β-D-maltoside. Complex I activity is measured in the presence of 2.5 mg · ml⁻¹ bovine serum albumin, which increases rotenone sensitivity, and we have shown that NADH-cytochrome b₅ reductase makes an important contribution to the rotenone-insensitive NADH-ubiquinone oxidoreductase activity. Complex II activity is measured after preincubation of the mitochondrial fraction with succinate to fully activate the complex. Complex I and III activities are dependent upon the length of the isoprenoid chain of the ubiquinone and ubiquinol, respectively. These assays have been used to establish a control range.     

Architecture of active mammalian respiratory chain supercomplexes

In the inner mitochondrial membrane, the respiratory chain complexes generate an electrochemical proton gradient, which is utilized to synthesize most of the cellular ATP. According to an increasing number of biochemical studies, these complexes are assembled into supercomplexes. However, little is known about the architecture of the proposed multicomplex assemblies. Here, we report the electron microscopic characterization of the two respiratory chain supercomplexes I1III2 and I1III2IV1 in bovine heart mitochondria, which are also two major supercomplexes in human mitochondria. After purification and demonstration of enzymatic activity, their structures in projection were determined by single particle image analysis. A difference map between the supercomplexes I1III2 and I1III2IV1 closely fits the x-ray structure of monocomplex IV and shows its location in the assembly. By comparing different views of supercomplex I1III2IV1, the location and mutual arrangement of complex I and the complex III dimer are discussed. Detailed knowledge of the architecture of the active supercomplexes is a prerequisite for a deeper understanding of energy conversion by mitochondria in mammals.