共查询到20条相似文献,搜索用时 15 毫秒
1.
Bax and heart mitochondria: uncoupling and inhibition of respiration without permeability transition
Appaix F Guerrero K Rampal D Izikki M Kaambre T Sikk P Brdiczka D Riva-Lavieille C Olivares J Longuet M Antonsson B Saks VA 《Biochimica et biophysica acta》2002,1556(2-3):155-167
The effects of Bax (full-length, FL, and C-terminal truncated, DeltaC) on respiration rate, membrane potential, MgATPase activity and kinetics of regulation of respiration were studied in isolated rat heart mitochondria and permeabilized cardiomyocytes. The results showed that while both Bax-FL and Bax-DeltaC permeabilized the outer mitochondrial membrane, released cytochrome c and reduced the respiration rate, the latter could be fully restored by exogenous cytochrome c only in the case of Bax-DeltaC, but not in presence of Bax-FL. In addition, Bax-FL but not Bax-DeltaC increased the MgATPase activity, and their effects on the mitochondrial membrane potential were quantitatively different. None of these effects was sensitive to cyclosporin A (CsA).It is concluded that Bax-FL affects both the outer and the inner mitochondrial membranes by: (1) opening large pores in the outer membrane; (2) inhibiting some segments of the respiratory chain in the inner membrane; and (3) uncoupling the inner mitochondrial membrane by increasing proton leak without opening the permeability transition pore (PTP). 相似文献
2.
The osmotic basis of low and high amplitude swelling in mitochondria was investigated in detail using sucrose and mannitol as external osmolytes. Osmotic behaviour of mitochondria in various respiratory states was consistent with significant changes in the porosity of the inner membrane corresponding to the rate of respiration. The stoichiometry of oxidative phosphorylation was confirmed to be dependent on the physical state (i.e., osmotic stretch) of the inner membrane regardless of the external polyol used. High amplitude swelling in polyol media was shown to arise from a sequential disruption of the outer and inner mitochondrial membranes, due to a dynamic instability induced by a combination of respiration, unscreened (fixed) surface charge density and the consequent variable porosity of the inner membrane. These novel experimental findings based on the physical theory of osmosis emphasize the need to define the fine structural changes of the inner membrane associated with oxidative phosphorylation to arrive at a comprehensive mechanism. 相似文献
3.
F Di Jeso D Giorgini S Bartolucci 《Comptes rendus des séances de la Société de biologie et de ses filiales》1978,172(1):67-72
Specific antibody has been obtained against cytochrome b (pig heart mitochondria). It inhibits the electron transport of the respiratory chain in the intact mitochondria at the cytochrome b site of the inner mitochondrial membrane. It has no effect on the isolated submitochondrial particles which are inside-out inner membrane vescicles free of any outer membrane or outside-out inner membrane. These findings indicate a probably not transmembranous topologic localization of cytochrome b; this component of the respiratory chain seems located near the outer side of the inner mitochondrial membrane. 相似文献
4.
Kozlova MV Gramadskiĭ KB Solodovnikova IM Krasinskaia IP Vinogradov AV Iaguzhinskiĭ LS 《Biofizika》2003,48(3):443-452
The study is devoted to the registration of local H+ gradients on the inner membrane of mitochondria under conditions of H+ pump functioning were recorded. By using a covalently linked pH probe (fluorescein isothiocyanate), a local increase in the activity of hydrogen ions on the outer face of the inner mitochondrial membrane in the presence of the respiration substrate at increased permeability of the membrane for K+ was registered. It was also found that the buffer capacity of medium affects the respiration rate of completely uncoupled mitochondria; a change in respiration rate strictly correlates with changes in local H+ gradients on the mitochondrial membrane. It was concluded that local gradients of H+ activity can control the rate of functioning of H+ pumps. It was shown that, under certain conditions, the system of H+ pumps incorporated into succinate oxidase of mitochondria functions as a nonliner system. 相似文献
5.
H Zaluska J Brabcová A Wroniszewska J Zborowski Z Drahota L Wojtczak 《Experimental cell research》1975,91(1):63-72
Digitonin treatment and the swelling-shrinkage-sonication procedure as used to separate mitochondria membranes were applied to mitochondria from the brown adipose tissue (BAT) of infant rats. Digitonin at a concentration of 0.15 mg/mg mitochondrial protein produced disruption of the outer membrane of BAT mitochondria and a complete release of adenylate kinase. However, fragments of the outer membrane remained firmly attached to the inner membrane-matrix particles (mitoplasts) and sedimented at 10 000 g, as indicated by the activity of monoamine oxidase in the pellet. Only at 0.5 mg digitonin/mg protein did outer membrane become almost entirely separated. Oxidation of external cytochrome c by mitoplasts was only 50% of the total cytochrome oxidase at 0.5 mg digitonin/mg protein, indicating an incomplete exposure of the inner membrane to the external medium. Ultrastructural studies revealed that a large proportion of mitoplasts retained the orthodox configuration under these conditions. Outer membrane fragments obtained by the swelling-shrinkage-sonication procedure were of buoyant density corresponding to 20–30% (weight/vol) sucrose. After a 10 sec sonication of mitochondria, a relatively pure outer membrane fraction could be obtained with a yield not exceeding 20%. Longer sonication increased the yield, but also increased the degree of contamination by inner membrane fragments. Optimum conditions for the separation of outer and inner membranes from brown adipose tissue mitochondria are described. 相似文献
6.
F di Jeso S Bartolucci D Giorgini 《Comptes rendus des séances de la Société de biologie et de ses filiales》1978,172(2):274-277
A specific antibody against cytochrome c1 (pig heart mitochondria) has been obtained. It inhibits the electron transport of the respiratory chain in the intact mitochondria at the cytochrome c1 site of inner mitochondrial membrane ; but it has no effect on the isolated submitochondrial particles (inside-out inner mitochondrial membrane vesicles free of any outer membrane or outside-out inner membrane). Thus the topologic position of cytochrome c1 in the inner mitochondrial membrane is asymetrically lcoated on the outer side of the inner mitochondrial membrane. These results agree with our previous researches on ATP-ase and cytochromes b, c and a, indicating the location on the inner side for the first one, transmembranous for the last one, on the outer side for the others respiratory chain components. Thus the electron transport from cytochrome b to a takes place in the outer region of inner mitochondrial membrane and the transmembranous location of cytochrome-oxidase facilitates the transfer of the electrons to oxygen. 相似文献
7.
A study has been carried out on the interaction of arachidonic acid and other long chain free fatty acids with bovine heart mitochondria. It is shown that arachidonic acid causes an uncoupling effect under state 4 respiration of intact mitochondria as well as a marked inhibition of uncoupled respiration. While, under our conditions, the uncoupling effect is independent of the fatty acid species considered, the inhibition is stronger for unsaturated acids. Experiments carried out with mitochondrial particles indicated that the arachidonic acid dependent decrease of the respiratory activity is caused by a selective inhibition of Complex I and III. It is also shown that arachidonic acid causes a remarkable increase of hydrogen peroxide production when added to mitochondria respiring with either pyruvate+malate or succinate as substrate. The production of reactive oxygen species (ROS) at the coupling site II was almost double than that at site I. The results obtained are discussed with regard to the impairment of the mitochondrial respiratory activity as occurring during the heart ischemia/reperfusion process. 相似文献
8.
NONDROPLET ULTRASTRUCTURAL DEMONSTRATION OF CYTOCHROME OXIDASE ACTIVITY WITH A POLYMERIZING OSMIOPHILIC REAGENT, DIAMINOBENZIDINE (DAB) 总被引:52,自引:12,他引:40
下载免费PDF全文
![点击此处可从《The Journal of cell biology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Arnold M. Seligman Morris J. Karnovsky Hannah L. Wasserkrug Jacob S. Hanker 《The Journal of cell biology》1968,38(1):1-14
A new method for demonstrating cytochrome oxidase activity, based upon the oxidative polymerization of 3,3'-diaminobenzidine (DAB) to an osmiophilic reaction product, has improved the localization of this enzyme over methods based upon the Nadi reaction, in both the light and electron microscopes. The reaction product occurs in nondroplet form, which more accurately delineates the localization of cytochrome oxidase in mitochondria of heart, liver, and kidney. In electron microscopic preparations the excess reaction product is found to overflow into the intracristate spaces and into the outer compartment between inner and outer limiting mitochondrial membranes. This finding suggests that the enzymatic activity of cytochrome c is located on the inner surface of the intracristate space which is the outer surface of the inner mitochondrial membrane. Succinic dehydrogenase activity has also been located at this site by using an osmiophilic ditetrazolium salt, TC-NBT. Considered together, the sites of reactivity of both parts of the respiratory chain have implications for the chemiosomotic hypothesis of Mitchell who suggests a mechanism of energy conservation during electron transport in the respiratory chain of the mitochondrion. 相似文献
9.
Highly purified mitochondria from rat liver were separated into six sub-fractions by differential centrifugation. The sub-fractions represent a spectrum from “heavy” to “very light” mitochondria. Enzymes representative of mitochondrial compartments were assayed to see whether functional differences occurred among the various mitochondrial sub-fractions. Respiratory control and NADH oxidase activity, both of which are indicators of mitochondrial structural integrity, were also measured. An enzyme marker for endoplasmic reticulum (glucose-6-phosphatase, G-6-Pase) was also assayed. Specific activities for monoamine oxidase (outer membrane marker), cytochrome oxidase (inner membrane marker) and malate-cytochrome c reductase did not vary within experimental error in all sub-fractions; similarly, for respiratory control and NADH oxidase activity. Malate dehydrogenase, a component of malate-cytochrome c reductase is located within the matrix surrounded by the inner membrane. Specific activity of adenylate kinase (located between the outer and inner membrane) decreased markedly from the “heavy” mitochondria to the “very light” fractions. Specific activity for G-6-Pase, very low in the “heavy” fractions, increased markedly in the “light” to “very light” fractions. Isopycnic density centrifugation on a linear sucrose density gradient of each of the fractions indicated that the correlation coefficient for the sucrose concentrations at which cytochrome oxidase and G-6-Pase activities peaked was 0.995. Thus the “light” to “very light” mitochondria may represent mitochondria whose outer membrane is still contiguous with the endoplasmic reticulum. Microsomes containing the endoplasmic reticulum peaked on the gradient at a significantly lower sucrose concentration than any of the mitochondrial sub-fractions. A buoyant effect of endoplasmic reticulum still attached to any of the mitochondrial sub-fractions would be expected to lower the density of attached mitochondria and thus give rise to “light” and “very light” mitochondria. 相似文献
10.
K Nicolay R J Timmers E Spoelstra R Van der Neut J J Fok Y M Huigen A J Verkleij B De Kruijff 《Biochimica et biophysica acta》1984,778(2):359-371
The interaction of adriamycin with cardiolipin in model membranes and in various membrane preparations derived from rat liver mitochondria was studied and the results are analyzed in the light of a possible specific interaction between adriamycin and cardiolipin. It was found that adriamycin binds to cardiolipin-containing model membranes with a fixed stoichiometry of two drug molecules per cardiolipin. Furthermore, the extent of drug complexation by mitochondria and mitoplasts (inner membrane plus matrix) is in reasonable agreement with their cardiolipin content. In contrast, adriamycin-binding curves of inner membrane ghosts and submitochondrial particles reveal considerable association to an additional site, presumably RNA. The evidence for the potential importance of RNA as a target comes from experiments on outer membranes and microsomes which both appear to bind substantial amounts of adriamycin. Removal of the major part of the RNA associated with these fractions by EDTA treatment is accompanied by a dramatic reduction of binding capacity. We propose that endogenous RNA present in mitochondria and mitoplasts is not accessible for adriamycin at low concentrations of the drug due to the presence of an intact lipid barrier. This potential site comes to expression in ghosts and submitochondrial particles, due to the absence of an intact lipid bilayer and due to the inside-out orientation of the limiting membrane, respectively. Electron microscopical studies show that adriamycin induces dramatic changes in mitochondrial morphology, similar to the uncoupler-induced effects described by Knoll and Brdiczka (Biochim. Biophys. Acta 733, 102-110 (1983). Adriamycin has an uncoupling effect on mitochondrial respiration and oxidative phosphorylation. The concentration dependence of this effect correlates with the adriamycin-binding curve for mitochondria which implies that only bound adriamycin actively inhibits respiration. 相似文献
11.
Evidence has been obtained for the occurrence of a cAMP-dependent serine protein kinase associated with the inner membrane/matrix of mammalian mitochondria. The catalytic site of this kinase is localized at the inner side of the inner membrane, where it phosphorylates a number of mitochondrial proteins. One of these has been identified as the AQDQ subunit of complex I. cAMP-dependent phosphorylation of this protein promotes the activity of complex I and mitochondrial respiration. A 5 bp duplication in the nuclear gene encoding this protein has been found in a human patient, which eliminates the phosphorylation site. PKA anchoring proteins have recently been identified in the outer membrane of mammalian mitochondria, which could direct phosphorylation of proteins at contact sites with other cell structures. 相似文献
12.
A Zó?kiewska B Zab?ocka J Duszyński L Wojtczak 《Archives of biochemistry and biophysics》1989,275(2):580-590
Rat liver mitochondria respiring under resting state conditions in the presence of oligomycin were rapidly blocked with cyanide and the dissipation of the membrane potential, measured with a tetraphenylphosphonium-sensitive electrode, was followed over time. The plot of the rate of membrane potential dissipation versus the actual value of the membrane potential was nonlinear and identical to the plot of resting state respiration (titrated with small amounts of a respiratory inhibitor) versus the membrane potential. The relationship between the respiratory chain activity and the proton-motive force in mitochondria oxidizing succinate with either oxygen or ferricyanide as electron acceptors was also found to be identical. These results are interpreted as an indication that the passive permeability of the inner mitochondrial membrane toward ions is far more significant in maintaining resting state respiration than is the molecular slippage of the pumps in the respiratory chain. These results also confirm the non-ohmic characteristics of the inner mitochondrial membrane. 相似文献
13.
Translocation and insertion of precursor proteins into isolated outer membranes of mitochondria 总被引:19,自引:3,他引:16
下载免费PDF全文
![点击此处可从《The Journal of cell biology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
《The Journal of cell biology》1993,121(6):1233-1243
Nuclear-encoded proteins destined for mitochondria must cross the outer or both outer and inner membranes to reach their final sub- mitochondrial locations. While the inner membrane can translocate preproteins by itself, it is not known whether the outer membrane also contains an endogenous protein translocation activity which can function independently of the inner membrane. To selectively study the protein transport into and across the outer membrane of Neurospora crassa mitochondria, outer membrane vesicles were isolated which were sealed, in a right-side-out orientation, and virtually free of inner membranes. The vesicles were functional in the insertion and assembly of various outer membrane proteins such as porin, MOM19, and MOM22. Like with intact mitochondria, import into isolated outer membranes was dependent on protease-sensitive surface receptors and led to correct folding and membrane integration. The vesicles were also capable of importing a peripheral component of the inner membrane, cytochrome c heme lyase (CCHL), in a receptor-dependent fashion. Thus, the protein translocation machinery of the outer mitochondrial membrane can function as an independent entity which recognizes, inserts, and translocates mitochondrial preproteins of the outer membrane and the intermembrane space. In contrast, proteins which have to be translocated into or across the inner membrane were only specifically bound to the vesicles, but not imported. This suggests that transport of such proteins involves the participation of components of the intermembrane space and/or the inner membrane, and that in these cases the outer membrane translocation machinery has to act in concert with that of the inner membrane. 相似文献
14.
N E Lofrumento D Marzulli L Cafagno G La Piana T Cipriani 《Archives of biochemistry and biophysics》1991,288(1):293-301
Oxidation of exogenous NADH by isolated rat liver mitochondria is generally accepted to be mediated by endogenous cytochrome c which shuttles electrons from the outer to the inner mitochondrial membrane. More recently it has been suggested that, in the presence of added cytochrome c, NADH oxidation is carried out exclusively by the cytochrome oxidase of broken or damaged mitochondria. Here we show that electrons can be transferred in and out of intact mitochondria. It is proposed that at the contact sites between the inner and the outer membrane, a "bi-trans-membrane" electron transport chain is present. The pathway, consisting of Complex III, NADH-b5 reductase, exogenous cytochrome c and cytochrome oxidase, can channel electrons from the external face of the outer membrane to the matrix face of the inner membrane and viceversa. The activity of the pathway is strictly dependent on both the activity of the respiratory chain and mitochondrion integrity. 相似文献
15.
Susanne E. Horvath Heike Rampelt Silke Oeljeklaus Bettina Warscheid Martin van der Laan Nikolaus Pfanner 《Protein science : a publication of the Protein Society》2015,24(3):277-297
Mitochondria import more than 1,000 different proteins from the cytosol. The proteins are synthesized as precursors on cytosolic ribosomes and are translocated by protein transport machineries of the mitochondrial membranes. Five main pathways for protein import into mitochondria have been identified. Most pathways use the translocase of the outer mitochondrial membrane (TOM) as the entry gate into mitochondria. Depending on specific signals contained in the precursors, the proteins are subsequently transferred to different intramitochondrial translocases. In this article, we discuss the connection between protein import and mitochondrial membrane architecture. Mitochondria possess two membranes. It is a long‐standing question how contact sites between outer and inner membranes are formed and which role the contact sites play in the translocation of precursor proteins. A major translocation contact site is formed between the TOM complex and the presequence translocase of the inner membrane (TIM23 complex), promoting transfer of presequence‐carrying preproteins to the mitochondrial inner membrane and matrix. Recent findings led to the identification of contact sites that involve the mitochondrial contact site and cristae organizing system (MICOS) of the inner membrane. MICOS plays a dual role. It is crucial for maintaining the inner membrane cristae architecture and forms contacts sites to the outer membrane that promote translocation of precursor proteins into the intermembrane space and outer membrane of mitochondria. The view is emerging that the mitochondrial protein translocases do not function as independent units, but are embedded in a network of interactions with machineries that control mitochondrial activity and architecture. 相似文献
16.
Changes in freeze-fractured mitochondrial membranes correlated to their energetic state. Dynamic interactions of the boundary membranes 总被引:6,自引:0,他引:6
Structural changes of mitochondria in correlation to their energetic state have been observed as matrix expansion and condensation. In this communication we describe a morphological correlation in freeze-fractured mitochondrial membranes which is also dependent on the metabolic state of the organelle: the frequency by which the fracture plane following the inner or outer boundary membrane deviates by jumping from one membrane to the other is higher in phosphorylating mitochondria when compared to freshly isolated or energized mitochondria. These deflections of the fracture plane occur mostly in minimal, short steps showing close apposition of the two boundary membranes. We therefore conclude that the observed change in morphological appearance is produced by a change in interactions between the inner and outer membranes correlated to the different functional states of the inner membrane. 相似文献
17.
Our aim was to carefully analyse the time-dependent changes that affect the mitochondrial function of myocardial cells during and after an ischemic episode. To this end, variables characterizing mitochondrial function have been evaluated on myocardial samples from isolated rat hearts subjected to different conditions of ischemia. The technique of permeabilized fibers was used in order to evaluate the mitochondrial function whilst retaining intracellular structure.The earliest alteration that could be detected was a decrease in the stimulatory effect of creatine on mitochondrial respiration. This alteration became more pronounced as the severity (or duration) of the ischemia increased. Afterwards, a significant decrease in the apparent Km of mitochondrial respiration for ADP also appeared, followed by a diminution of the maximal respiration rate which was partly restored by adding cytochrome c. Finally, for the most severe conditions of ischemia, the basal respiratory rate also increased. These observations are indicative of a sequence of alterations affecting first the intermembrane space, then the outer mitochondrial membrane, and finally the inner membrane. The discussion is focused on the very early alterations, that could not be detected using the conventional techniques of isolated mitochondria. We postulate that these alterations to the intermembrane space and outer mitochondrial membrane can induce disturbances both in the channelling of energy from the mitochondria, and on the signalling towards the mitochondria. The potential consequences on the regulation of the production of energy (ATP, PC) by the mitochondria are evoked. 相似文献
18.
Kicinska A Swida A Bednarczyk P Koszela-Piotrowska I Choma K Dolowy K Szewczyk A Jarmuszkiewicz W 《The Journal of biological chemistry》2007,282(24):17433-17441
We describe the existence of a potassium ion transport mechanism in the mitochondrial inner membrane of a lower eukaryotic organism, Acanthamoeba castellanii. We found that substances known to modulate potassium channel activity influenced the bioenergetics of A. castellanii mitochondria. In isolated mitochondria, the rate of resting respiration is increased by about 10% in response to potassium channel openers, i.e. diazoxide and BMS-191095, during succinate-, malate-, or NADH-sustained respiration. This effect is strictly dependent on the presence of potassium ions in an incubation medium and is reversed by glibenclamide (a potassium channel blocker). Diazoxide and BMS-191095 also caused a slight but statistically significant depolarization of mitochondrial membrane potential (measured with a TPP(+)-specific electrode), regardless of the respiratory substrate used. The resulting steady state value of membrane potential was restored after treatment with glibenclamide or 1 mM ATP. Additionally, the electrophysiological properties of potassium channels present in the A. castellanii inner mitochondrial membrane are described in the reconstituted system, using black lipid membranes. Conductance from 90 +/- 7 to 166 +/- 10 picosiemens, inhibition by 1 mM ATP/Mg(2+) or glibenclamide, and activation by diazoxide were observed. These results suggest that an ATP-sensitive potassium channel similar to that of mammalian mitochondria is present in A. castellanii mitochondria. 相似文献
19.
《Biochimica et Biophysica Acta (BBA)/Molecular and Cell Biology of Lipids》2017,1862(1):102-113
Mitochondria contain two membranes: the outer and inner membrane. Whereas the outer membrane is particularly enriched in phospholipids, the inner membrane has an unusual high protein content and forms large invaginations termed cristae. The proper phospholipid composition of the membranes is crucial for mitochondrial functions. Phospholipids affect activity, biogenesis and stability of protein complexes including protein translocases and respiratory chain supercomplexes. Negatively charged phospholipids such as cardiolipin are important for the architecture of the membranes and recruit soluble factors to the membranes to support mitochondrial dynamics. Thus, phospholipids not only form the hydrophobic core of biological membranes that surround mitochondria, but also create a specific environment to promote functions of various protein machineries. This article is part of a Special Issue entitled: Lipids of Mitochondria edited by Guenther Daum. 相似文献
20.
Membrane lipids of yeast mitochondria have been enriched by growing yeast cells in minimal medium supplemented with specific unsaturated fatty acids as the sole lipid supplement. Using the activity of marker enzymes for the outer (kynurenine hydroxylase) and inner (cytochrome c oxidase and oligomycin-sensitive ATPase) mitochondrial membranes, Arrhenius plots have been constructed using both promitochondria and mitochondria obtained from O2-adapting cells in the presence of a second unsaturated fatty acid (i.e. linoleate (N2) to elaidic (O2)). Transition temperatures which reflect the unsaturated fatty acid enrichment of the new membranes reveal interesting features involved in the mechanism of the assembly of these two mitochondrial membranes. This approach was further enforced with both lipid depletion and mitochondrial protein inhibition studies. Kynurenine hydroxylase which does not require fatty acid for its continued synthesis during aerobiosis seems to be incorporated into the preformed linoleate-anaerobic outer membrane. The newly synthesized activities of inner mitochondrial membrane enzymes on the other hand, appear to integrate their activity into newly formed aerobic-elaidic-rich inner membrane. These latter enzymes show a distinct dependence on fatty acid supplement for their continued synthesis during their aerobic phase. This suggests that O2-dependent proteo-lipid precursors are formed before these enzymes are integrated into their membrane mosaic. Two separate models are proposed to explain these results, one for the lipid-rich outer mitochondrial membrane and another for the protein-rich inner mitochondrial membrane. 相似文献