Action of propranolol on mitochondrial proton fluxes.

The action of propranolol, a Beta-blocking adrenergic agent, on mitochondrial proton fluxes in nonenergized and energized conditions has been studied. 1. The drug inhibited the actions of valinomycin, higericin and FCCP on the inner mitochondrial membrane. 2. It decreased the rate and extent of active energized proton expulsion and passive collapse of the proton gradient so formed. 3. Propranolol was able to increase the permeation of chloride ion through the inner mitochondrial membrane in nonenergized and energized conditions. 4. The drug inhibited mitochondrial contraction but stimulated swelling in various conditions. It is suggested that propranolol is is able to change the proton and chloride permeabilities of mitochondria by perturbing the structure of inner membrane phospholipids, thus enlarging the water-lipid interface.     

Investigations of mitochondrial adenine nucleotide translocation by means of nucleotide analogs.

ATP and ADP are transferred across the inner mitochondrial membrane by means of a carrier (translocator), an inner membrane integral lipoprotein. Translocation of the adenine nucleotides occurs in two steps: specific binding and transport. By using substrate analogs with modified adenine, phosphate, or ribose moieties it is possible to check which structural properties of the substrate are essential for binding and transport.     

Electrostatic interactions between model mitochondrial membranes

Lipids are very diverse in both their respective structures and functions; and cells exquisitely control membrane composition. One intriguing issue is the specific role of lipids in modulating the physical properties of membranes. Cardiolipin (CL) is a unique four-tailed, doubly negatively charged lipid found predominately within the inner mitochondrial membrane, and is thought to be influential in determining the inner mitochondrial membrane potential and permeability. To determine the role of cardiolipin in modulating the charge properties of membranes, this study investigated the electrostatic interactions between mixed cardiolipin and phosphatidylcholine bilayers as a function of cardiolipin concentration. For physiologically relevant concentrations of cardiolipin, the surface charge density of the membrane was found to increase linearly with increasing concentration of cardiolipin. However, only a fraction of the cardiolipin molecules predicted to carry a charge from pK-values were ionized. Clearly environmental factors, beyond that of pH, play a role in determining the charge of bilayers containing cardiolipin.     

Possible involvement of mitochondrial calcium transport in causing cell injury in experimental hepatic chronic iron overload

The functional state of isolated mitochondria and, specifically, the integrity of the inner membrane were investigated in the livers of rats made siderotic by dietary supplementation with carbonyl iron. The concentration of iron in the mitochondrial fraction increased progressively up to nearly 40 days and it reached a steady-state level. When the iron content reached a threshold value (higher than 30 nmol/mg protein) the occurrence of in vivo lipid peroxidation in the mitochondrial membrane was detected. This process did not result in gross alterations in the mitochondrial membrane, as indicated by phosphorylative capability and membrane potential measurements. On the contrary, the induction of the lipoperoxidative reaction appeared to be associated with the activation of Ca²⁺ release from mitochondria. This was shown to occur as a consequence of rather subtle modifications in the inner membrane structure via a specific efflux route, which appeared to be linked to the oxidation level of mitochondrial pyridine nucleotides. The induction of this Ca²⁺ release from iron treated mitochondria resulted in enhancement of Ca²⁺ cycling, a process which dissipates energy to reaccumulate the releaased Ca²⁺ into mitochondria. The perturbation in mitochondrial Ca²⁺ homeostasis reported here may be a factor in the onset of cell damage in this experimental model of hepatic chronic iron overload.     

Mitochondria-penetrating peptides

Mitochondria are important targets for cancer chemotherapy and other disease treatments. Gaining access to this organelle can be difficult, as the inner membrane is a barrier limiting diffusive transport. A mitochondrial molecular carrier would be a boon to the development of organelle-specific therapeutics. Here, we report a significant advance in the development of mitochondrial transporters-synthetic cell-permeable peptides that are able to enter mitochondria. Efficient uptake of these mitochondria-penetrating peptides (MPPs) is observed in a variety of cell types, and organellar specificity is attained with sequences that possess specific chemical properties. The MPPs identified are cationic, but also lipophilic; this combination of characteristics facilitates permeation of the hydrophobic mitochondrial membrane. The examination of a panel of MPPs illustrates that mitochondrial localization can be rationally controlled and finely tuned by altering lipophilicity and charge.     

可固定性荧光探针:线粒体成像的可靠工具

线粒体是一种具有双层膜结构的细胞器,参与细胞新陈代谢过程的能量循环以及离子平衡过程,在细胞生理过程中具有极其重要的意义。一些小分子荧光染料/探针结构中带有正电荷,因受到线粒体内膜负电势的牵引而标记在线粒体上,为研究线粒体的形态或功能提供了重要的可视化成像工具。然而,大多数线粒体染料/探针对线粒体的靶向标记稳定性仍不够理想,因为线粒体电势处于不断的动态变化中,当电势降低时,对染料的亲和力相应降低。尤其在病理条件下(比如细胞凋亡)细胞代谢受到阻滞时,线粒体膜电势显著降低,阳离子染料将扩散离开线粒体,造成非特异性荧光。最近,Kim团队和本人课题组提出可固定线粒体探针的新概念,用活性基团将荧光分子探针通过共价键固定在线粒体中,开发了稳定靶向线粒体中的定量探测微环境p H值、粘度、膜电势荧光探针。我们认为,对于追踪和探测具有高度动态变化特性的线粒体而言,开发可固定的线粒体荧光分子探针是必然趋势,因此本文进行评述和展望。     

Capillary electrophoresis of cardiolipin with on-line dye interaction and spectrophotometric detection

Cardiolipin is an important phospholipid present in the mitochondrial inner membrane. It plays a key function in mitochondrial respiration by interacting with many enzymes or cofactors related to oxidative phosphorylation complexes. We have determined the concentration of cardiolipin using on-line 10-N-nonyl acridine orange (NAO) dye interaction capillary electrophoresis (CE) and spectrophotometric detection with a sample throughput of 3 min. In addition to the presence of 0.1 mM NAO, the background electrolyte (BGE) composition has been set at 80% methanol-10% acetonitrile-10% H(2)O (all v/v) to provide both good solubility and the maximum absorbance enhancement at 497 nm for the NAO-cardiolipin complex as compared to NAO alone. Sample consumption for each injection is about 57 nL. A calibration curve is established from 0.5 microM to 0.1 mM with R (2) = 0.9912 with a detection limit of 0.05 microM for cardiolipin. In a blind study, actual mitochondrial cell membrane samples in the microL range before or after UV light exposure were analyzed using the CE method. Cardiolipin concentration decreased in the different parts of the membrane sample upon UV photolysis of the cells. Support for the theory that UV light can induce cardiolipin translocation from the inner membrane (IM) to the outer membrane (OM) was indicated by a significant percentage increase of cardiolipin (as measured by the cardiolipin in the OM as compared to the sum total in the OM and IM) from 30.7 +/- 2.4% before UV light photolysis to 38.3 +/- 2.2% after UV irradiation.     

Energy transfer microscopy for probing mitochondrial deficiencies

The autofluorescence of intact and respiratory deficiency yeast strains was measured using advanced microscopic techniques. Deficiencies in the inner mitochondrial membrane were correlated with an increase in NADH and flavin fluorescence. Since mitochondrial fluorescence was superposed by the luminescence of other intracellular sites, part of the cells were incubated with Rhondamine 123, a fluorescent mitochondrial marker. It could be shown that, depending on the excitation wavelength, Rhodamine 123 was excited either directly or by energy transfer from flavin molecules. The efficiency of this energy transfer may be used to probe the function of mitochondrial metabolism. Similarly, thionine and chloroaluminium phthalocyanine appear to be potential acceptors of excitation energy from mitochondrial cytochromes.     

Energy metabolism in mitochondria

Mitochondria are separate metabolic compartments within the cell. The functional boundary of the mitochondrial compartment is the inner membrane. This membrane contains the enzymatic apparatus for the electron transport and oxidative phosphorylation. The substrate breakdown cycles are localized in the mitochondrial matrix space. Specific carriers are responsible for the exchange of ADP, ATP, phosphate, and intermediates of the citric acid cycle between the matrix space and the extramitochondrial space. The particular importance of the adenine nucleotide transport to the regulation of the energy metabolism of the cell is discussed in detail.     

Mitochondrial Oxidative Phosphorylation

Mitochondria can form ATP from ADP and inorganic phosphate, the required energy being supplied by respiration. This coupled process, which in sufficiently aerated normal animal cells furnishes the bulk of the cellular ATP, is termed oxidative phosphorylation. The overall reaction is intimately associated with the mitochondrial inner membrane and proceeds via a primary high-energy intermediate. This intermediate, in a manner as yet unknown, energizes the anhydride formation between ADP and inorganic phosphate. The coupling between respiration and ATP synthesis is mediated by proteins of the mitochondrial inner membrane which are known as “coupling factors”. The mechanism of oxidative phosphorylation is at present being discussed in terms of three hypotheses which are generally referred to as “chemical”, “chemiosmotic”, and “conformational” hypotheses. None of these hypotheses has as yet been experimentally verified.     

Mitochondrial oxidative phosphorylation in autosomal dominant optic atrophy

Background  

Autosomal dominant optic atrophy (ADOA), a form of progressive bilateral blindness due to loss of retinal ganglion cells and optic nerve deterioration, arises predominantly from mutations in the nuclear gene for the mitochondrial GTPase, OPA1. OPA1 localizes to mitochondrial cristae in the inner membrane where electron transport chain complexes are enriched. While OPA1 has been characterized for its role in mitochondrial cristae structure and organelle fusion, possible effects of OPA1 on mitochondrial function have not been determined.     

Molecular mechanism of thiamine pyrophosphate import into mitochondria: a molecular simulation study

The import of thiamine pyrophosphate (TPP) through both mitochondrial membranes was studied using a total of 3-µs molecular dynamics simulations. Regarding the translocation through the mitochondrial outer membrane, our simulations support the conjecture that TPP uses the voltage-dependent anion channel, the major pore of this membrane, for its passage to the intermembrane space, as its transport presents significant analogies with that used by other metabolites previously studied, in particular with ATP. As far as passing through the mitochondrial inner membrane is concerned, our simulations show that the specific carrier of TPP has a single binding site that becomes accessible, through an alternating access mechanism. The preference of this transporter for TPP can be rationalized mainly by three residues located in the binding site that differ from those identified in the ATP/ADP carrier, the most studied member of the mitochondrial carrier family. The simulated transport mechanism of TPP highlights the essential role, at the energetic level, of the contributions coming from the formation and breakage of two networks of salt bridges, one on the side of the matrix and the other on the side of the intermembrane space, as well as the interactions, mainly of an ionic nature, formed by TPP upon its binding. The energy contribution provided by the cytosolic network establishes a lower barrier than that of the matrix network, which can be explained by the lower interaction energy of TPP on the matrix side or possibly a uniport activity.

     

Identification of novel anti cancer agents by applying insilico methods for inhibition of TSPO protein

Cancer is a genomic disease characterised as impaired cellular energy metabolism. Cancer cells derive most of their energy from oxidative phosphorylation unlike normal ones during cell progression TSPO protein present in external mitochondrial membrane, is involved in various cellular functions like Cell proliferation, mitochondrial respiration, synthesis of steroids and also participates in import of cholesterol into the inner mitochondrial membrane from outside of the membrane of mitochondria.The 3D model of TSPO protein is built using comparative homology modelling techniques and validated by proSA, Ramachandran plot and ERRAT in the present work. Active site prediction is carried out using SiteMap and literature, which allows the prediction of the important binding pockets for the identification of putative active site. New molecular entities as TSPO inhibitors were obtained from Virtual screening using MS Spectrum databank in Schrodinger suite and were prioritised based on Glide Score. Docking was performed using Autodock to identify molecules with different scaffolds and were prioritised based on binding energy and RMSD values. Qikprop is used to calculate pharmacokinetic properties of the screened molecules which are found to be in permissible range as possible novel inhibitors of TSPO protein to supress cell proliferation.     

A coulombic hypothesis of mitochondrial energy transduction

A coulombic (electrostatic) interpretation of mitochondrial energy transduction is proposed as an alternative to the chemiosmotic (electrochemical) view. Both the hypotheses accept the importance of proton translocation across the inner membrane as a primary consequence of electron transport; only the coulombic hypothesis gives significance to the conjoint phenomenon of negative fixed charge formation.     

Effect of La on heat production by mitochondria isolated from hybrid rice

The effect of lanthanum on mitochondria isolated from hybrid rice Fengyou 559 (Oryza sativa L.) was investigated. Through in vivo culture, low-dose La³⁺ promoted, but higher dose La³⁺, restrained mitochondrial heat production. However, through in vitro incubation, La³⁺ manifested only inhibitory action on mitochondrial energy turnover, the concentration required for 50% and 100% inhibition being 50.9 and 230.2 μM (57.6 nmol/mg protein), respectively. In addition, La³⁺, like Ca²⁺, induced rice mitochondrial swelling and decreased membrane potential (Δψ), which was inhibited by the specific permeability transition inhibitor cyclosporine A (CsA). The induction approached a constant limitation while mitochondrial metabolism was completely prevented by La³⁺, and microscopy observation showed a high disruption of inner mitochondrial membrane in this state. These results demonstrated that lanthanum influenced rice mitochondria in vivo and in vitro via different action pathways, and the latter involved the opening of rice mitochondrial permeability.     

Comparison of N-alkyl acridine orange dyes as fluorescence probes for the determination of cardiolipin

The phospholipid (PL), cardiolipin (CL), is found almost exclusively in the inner membrane of mitochondria and loss of CL is considered as an important indication of cell apoptosis. Previously, 10-N-nonyl acridine orange (NAO) has been used as a fluorescent probe for the visualization of CL in mitochondrial cell membranes and in solution. In this work for the determination of CL, we have synthesized two new fluorescent probes, n-tetradecyl acridine orange (C14-AO), and n-octadecyl acridine orange (C18-AO) by reacting acridine orange with the corresponding n-alkyl bromide. Using excitation and emission wavelengths at about 500 and 525 nm and varying the percentage of methanol in water as the solvent, no interaction between CL and the fluorescent probes at 75% is noted but a proportional quenching of the fluorescence signal by CL is observed at 50% or less for C14-AO and 60% or less for C18-AO. Binding efficiency of these fluorescent probes to CL is compared using dye concentrations of 5, 10, and 20 μM. C18-AO shows a better sensitivity than C14-AO and NAO, respectively, but is less selective. For C14-AO, the detection limit and limit of quantitation are 0.07 and 0.21 μM, respectively, which are better than those previously reported for NAO. One anionic PL, phosphatidic acid, shows some quenching interference to both the C14 and C18 dyes but only at concentrations above the working range for sample analysis. The CL in mitochondrial membrane samples is determined by standard addition using C14-AO. The level of CL in the outer mitochondrial membrane compared to the inner membrane is significantly increased due to the addition of cadmium chloride into the cells causing cell apoptosis.     

Aryl urea substituted fatty acids: a new class of protonophoric mitochondrial uncoupler that utilises a synthetic anion transporter

Respiring mitochondria establish a proton gradient across the mitochondrial inner membrane (MIM) that is used to generate ATP. Protein-independent mitochondrial uncouplers collapse the proton gradient and disrupt ATP production by shuttling protons back across the MIM in a protonophoric cycle. Continued cycling relies on the formation of MIM-permeable anionic species that can return to the intermembrane space after deprotonation in the mitochondrial matrix. Previously described protonophores contain acidic groups that are part of delocalised π-systems that provide large surfaces for charge delocalisation and facilitate anion permeation across the MIM. Here we present a new class of protonophoric uncoupler based on aryl-urea substituted fatty acids in which an acidic group and a π-system are separated by a long alkyl chain. The aryl-urea group in these molecules acts as a synthetic anion receptor that forms intermolecular hydrogen bonds with the fatty acid carboxylate after deprotonation. Dispersal of the negative charge across the aryl-urea system produces lipophilic dimeric complexes that can permeate the MIM and facilitate repeated cycling. Substitution of the aryl-urea group with lipophilic electron withdrawing groups is critical to complex lipophilicity and uncoupling activity. The aryl-urea substituted fatty acids represent the first biological example of mitochondrial uncoupling mediated by the interaction of a fatty acid and an anion receptor moiety, via self-assembly.

A new mitochondrial uncoupler that forms membrane permeable dimers through interactions of remote acidic and anion receptor groups.     

Subcomplexes of mitochondrial complex V reveal mutations in mitochondrial DNA

Complex V, site of the final step in oxidative phosphorylation, uses the proton gradient across the inner mitochondrial membrane for the production of ATP. It is a multi‐subunit complex composed of a catalytic domain (F₁) and a membrane domain (F₀) linked by two stalks. Subcomplexes of complex V containing the F₁ domain have previously been reported in small series of patients. We report the results in tissue samples and/or cultured skin fibroblasts studied by blue native PAGE followed by activity staining in the gel. Catalytically active subcomplexes of complex V were detected in 66 tissues originating from 53 patients. In 29 of the latter (55%), a mitochondrial DNA (mtDNA) defect was identified. Twelve patients had a pathogenic point mutation in a mitochondrial tRNA, one a large mtDNA deletion, 12 showed mtDNA depletion and four had a mutation in the MT‐ATP6 gene. We conclude that the presence of subcomplexes of complex V is a valuable indicator in the detection of mtDNA defects.     

Die zitterfreie Thermogenese: Wärmeproduktion bei Kältestress

Non‐shivering thermogenesis is based on the increase of the permeability of the inner membrane of mitochondria to H⁺. An increase in the H⁺‐flux from the cytosolic to the matrix compartiment dissipates the electrochemical proton gradient and, therefore, activates the mitochondrial respiratory chain. Permeability of H⁺ is stimulated by artificial protonophores such as 2,4‐dinitrophenol or the interaction of non‐esterified fatty acids with certain proteins of the inner membrane of mitochondria, called uncoupling proteins (UCPs). UCPs are expressed in various tissues of mammalians, but also in riping plants. Despite of their role in thermogenesis, activated UCPs suppress the formation of the superoxid anion radical. Therefore, UCPs are also physiological tools to diminish oxidative stress within the cell.