Membrane Of Submitochondrial Particles Research Articles

Bcl-xL Determines the Metabolic Efficiency of Neurons, through Interaction with Mitochondrial ATP Synthase

The anti-apoptotic BCL-2 family protein, Bcl-xL, is highly expressed in the brain. Beside its anti-apoptotic function, Bcl-xL may contribute to activity-dependent normal functioning of neurons. In this study, using a set of gain and loss of function experiments, we find that, in hippocampal neurons, Bcl-xL enhances the efficiency of neuronal energy metabolism by increasing total cellular ATP levels, while decreasing cellular oxygen use. This effect is due to the presence of Bcl-xL in the mitochondrial inner membrane, co-localized with the (F1F0) ATP synthase, shown by immunocytochemistry and immune-electron microscopy. The interaction causes a decrease in leak of H+ ions across the mitochondrial inner membrane. The decreased leak is correlated with an increase in coupling of oxidative phosphorylation. In contrast, inhibition of Bcl-xL by pharmacological or genetic means increases a leak conductance measured by patch clamping the membrane of submitochondrial particles enriched in ATP synthase complexes (SMVs). Leaky SMVs demonstrate attenuated ability to sequester H+ during ATP hydrolysis. Additionally, Bcl-xL protein directly increases ATPase activity of the purified synthase complexes, while inhibition of endogenous Bcl-xL decreases synthase activity. The exact site of the leak is being determined by cross-linking studies and by patch clamping individual protein components of the ATP synthase reconstituted into artificial lipid vesicles. Improved mitochondrial metabolic efficiency of neurons accompanying changes in leak level may result in long lasting changes in synaptic efficacy in both physiological and pathological conditions.

Translocation of Loops Regulates Transport Activity of Mitochondrial ADP/ATP Carrier Deduced from Formation of a Specific Intermolecular Disulfide Bridge Catalyzed by Copper-o-Phenanthroline

The cross-linking reagent copper-o-phenanthroline complex (Cu(OP)2) specifically caused a decrease in the amount of the 30-kDa ADP/ATP carrier in bovine submitochondrial particles associated predominantly with formation of a 60-kDa protein consisting of a cross-linked dimer of the carrier. However, Cu(OP)2 had no effect on mitochondria. The transport of ADP via the carrier through submitochondrial particle membranes was found to be inhibited in parallel with the progress of intermolecular cross-linking. Analysis of the cross-linked site showed that a disulfide bridge was formed only between two Cys56 residues in a pair of the first loops facing the matrix space. The transport inhibitor bongkrekic acid, which locks the m-state conformation of the carrier, had no effect on disulfide bridge formation catalyzed by Cu(OP)2, but carboxyatractyloside, which locks the c-state conformation by acting from the cytosolic side, completely inhibited the cross-linking. These results show that the ADP/ATP carrier functions as a dimer form, and a pair of the first loops protrudes into the matrix space in the m-state, but possibly intrudes into the membrane in the c-state. Thus, it is suggested that a pair of the first loops acts as a gate and that its opening and closing are regulated by their translocation.

Mitochondrial electron transport-linked tocopheroxyl radical reduction

alpha-Tocopherol (vitamin E) is a lipophilic chain-breaking antioxidant which inhibits lipid peroxidation in isolated mitochondrial membranes and protects membranes from oxidative damage. The primary oxidation product of vitamin E is the tocopheroxyl radical. Reduction of the tocopheroxyl radical can occur by reactions with water-soluble anti-oxidants such as ascorbate or glutathione, resulting in the recycling of vitamin E. Physiological concentrations of vitamin E are too low to allow detection of tocopheroxyl radical by ESR. After dietary supplementation with vitamin E, a 10-20-fold increase in the rat liver mitochondrial membrane content of vitamin E was achieved and this allowed for direct detection of the tocopheroxyl radical by ESR, after treatment with an oxidizing system composed of lipoxygenase and arachidonic acid. By using submitochondrial particle membranes, it was shown that NADH, succinate, and reduced cytochrome c-linked oxidation reduce the tocopheroxyl radical, preventing both accumulation of the radical and vitamin E consumption. As the electron transport chain can reduce tocopheroxyl radical it may have an important physiological role in recycling vitamin E.

Chymotrypsin activates cardiac mitochondrial carnitine-acylcarnitine translocase

The carnitine-acylcarnitine translocase facilitates carnitine and acylcarnitine transport into the mitochondrial matrix during beta-oxidation. Our results demonstrate that chymotrypsin can activate the maximal velocity of N-ethylmaleimide (NEM)-sensitive carnitine or palmitoylcarnitine exchange 7-fold, while doubling the affinity of the translocase for carnitine. Chymotrypsin activation is strictly dependent on the presence of free or short-chain acylcarnitine in the proteolysis medium, the extent of activation decreasing as the acylcarnitine chain length in the proteolysis medium increases. Chymotrypsin treatment decreases the apparent I50 value (inhibitor concentration required to give half-maximal inhibition) of the translocase for inhibition by NEM only under conditions which produce translocase activation. Modification of submitochondrial particle membranes by chymotrypsin does not result in gross ultrastructural changes or in an increase in the passive permeability of these membranes to carnitine. The data suggest that carnitine binding produces a change in translocase conformation which allows chymotrypsin modification to occur. This modification alters the kinetic and inhibitor-binding properties of the translocase.

Free-radical-mediated fragmentation of monoamine oxidase in the mitochondrial membrane. Roles for lipid radicals.

A flux of hydroxyl radicals generated by gamma-irradiation can fragment monoamine oxidase in the membrane of submitochondrial particles. This fragmentation can be inhibited by mannitol and in addition is more extensive in monoamine oxidase preparations that have been depleted of lipid. This latter observation is consistent with the higher yields of fragmentation induced by hydroxyl radicals in soluble proteins in the absence of added lipids. In the absence of oxygen, gamma-irradiation of submitochondrial particles leads to cross-linking reactions. A flux of hydroperoxyl radicals also causes fragmentation, whereas one of superoxide is virtually inactive in this respect. The irradiation of submitochondrial particles leads in addition to the accumulation of products of lipid peroxidation. When these irradiated preparations are exposed to ferrous or cupric salts a further fragmentation of monoamine oxidase ensues, especially at acid pH. These transition-metal-catalysed reactions do not occur with irradiated preparations depleted of lipid, and the post-irradiation protein modifications are concomitant with further lipid peroxidation. The data indicate roles for lipid radicals in both fragmentation and cross-linking reactions of proteins in biological membranes. These reactions may have an important bearing on control of protein activity and of protein turnover in membranes.

Three proton pumps, morphology and movements.

The diameter of F1 coupling factor and the distance it protrudes from the membrane of bovine heart submitochondrial particles were measured quantitatively using horse spleen ferritin as a standard. Employing the freeze-etch technique, particles of similar size were found on membranes of submitochondrial particles and on membranes of particles first depleted by F1, then reconstituted by addition of F1. The extramembranous size of F1 is 9.7 nm and F1 protrudes from the membrane surface by about 13.6 nm. Bacteriorhodopsin and cytochrome oxidase were incorporated into lipids derived from membranes of extremely thermoacidophilic microorganisms by the octylglucoside dilution method. The bacteriorhodopsin pump was fully functional provided high concentrations of valinomycin were added. With decanoyl-N-methylglucamide as detergent the pump was very active in the absence of valinomycin. Concentrations of gramicidin that collapsed the delta pH in bacteriorhodopsin liposomes prepared with soybean phospholipid had little or no effect on these rigid proteoliposomes. Very high concentrations (30 micrograms per ml) were partially effective, suggesting a mechanism other than formation of a gramicidin dimer channel. Cytochrome oxidase lost virtually all activity when incorporated into these rigid liposomes but was fully reactivated on addition of suitable detergents.

Localization of a ferricyanide-reactive site of cytochrome b− c1 complex, possibly of cytochrome b or ubisemiquinone, at the outer face of submitochondrial particles

When succinate oxidation by submitochondrial particles is blocked by antimycin, NoHOQnO or funiculosin, addition of ferricyanide restores oxygen uptake coupled to membrane potential generation. The effect of ferricyanide is abolished by mucidin or myxothiazol, as well as by KCN. The data strongly favor a cyclic redox loop mechanism in site 2 and show that either heme of the ferrous cytochrome b or ubisemiquinone formed in the QH 2-oxidizing center of complex b− c 1 is accessible to ferricyanide at the outer (M) side of the submitochondrial particle membrane.

The interaction of the potential-sensitive molecular probe merocyanine 540 with phosphorylating beef heart submitochondrial particles under equilibrium and time-resolved conditions

The interaction of the potential-sensitive extrinsic molecular probe merocyanine 540 (M540) with phosphorylating submitochondrial particles has been investigated under equilibrium and time-resolved conditions. The addition of ATP to a M540-membrane suspension produces oligomycin and CCCP-sensitive spectral changes with absolute maxima near 490, 530, and 565 nm; a 1- to 2-nm red shift of the dye absorption spectrum is also evident in the longer-wavelength region of the spectrum. In fixed-wavelength work, the energy-dependent optical signals were increased by the addition of nigericin and NH 4Cl, and could be subsequently restored to the control level by valinomycin or KSCN, respectively. These observations suggest that M540 is specifically sensitive to the membrane-potential portion of the electrochemical gradient presumably present in the submitochondrial particle system in the presence of substrate. Binding analyses based on the Langmuir adsorption isotherm and the direct linear method indicate that the M540 dissociation constant is decreased by the presence of ATP with little or no change in the maximum number of binding sites. The M540 dissociation constant was markedly decreased when 0.1 m NaCl was present in the medium, suggesting that the association of this probe with the membrane may be subject to considerable surface charge repulsion. Results from the binding analyses indicate that the origin of the energy-dependent spectral changes may be an enhanced association of M540 with the submitochondrial particle membrane resulting from the transfer of dye from the aqueous phase to membrane-binding sites. The time course of the NADH-, ATP-, or succinate-induced signal developed slowly, on a time scale of tens of seconds, and follows a second-order rate law, suggesting that the rate-limiting step in the development of the ATP-induced M540 signal may be the transfer of dye from the aqueous phase to membranebinding sites. The enhanced passive binding of M540 to the submitochondrial particle membrane in the presence of NaCl reduces the concentration of free dye apparently available to redistribute to the membrane when substrate is present, with a concomitant reduction in the observed pseudo-first-order and the second-order rate constants. If the effective free dye concentration is estimated from binding data and used in the plot from which the latter rate constant is obtained, the value of this constant compares favorably with the obtained in the absence of the electrolyte. In a concentration range as large as 0 to 10 μ m, the presence of M540 in submitochondrial particles has no effect on the rate constants characterizing three energy-dependent processes: the ATP-induced cytochrome c oxidase soret band shift, and the oxidation of cytochrome 1 and the reduction of pyridine nucleotide, both via ATP-driven reversed electron transport. The failure of M540 to impede the rates at which these energy-intensive processes develop suggests that the M540 anion does not permeate the particle membrane in the time scale covered by these experiments, 0.3 s to some 15 min.

Subunit structure of submitochondrial particle membrane transhydrogenase.

The subunit structure of membrane-bound mitochondrial transhydrogenase was investigated. Chemical modification of bovine heart submitochondrial particles with the cleavable bifunctional cross-linking reagent, dithiobis(succinimidyl propionate), resulted in the formation of three dimeric "cross-link isomers" of the enzyme, identified by immunoautoradiography, that are characteristic of cross-linked purified transhydrogenase. A limited amount of cross-linking of transhydrogenase monomer to Mr = 25,000 polypeptide was also observed. At high concentration of the cross-linker, a small amount of a higher molecular weight species was formed with both purified and membrane enzyme. Reductive cleavage of the dimeric and higher molecular weight species resulted in the regeneration of transhydrogenase monomer and several other proteolytically derived fragments. It is concluded that transhydrogenase exists in the native membrane primarily as a dimeric species.

Solubilization and Partial Purification of N,N′-Dicyclohexylcarbodiimide-Sensitive ATPase from Pea Cotyledon Mitochondria

The N,N'-dicyclohexylcarbodiimide (DCCD)-sensitive ATPase of pea (Pisum sativum L.) cotyledon mitochondria was solubilized from submitochondrial particle membranes with sodium cholate and ammonium sulfate. Ammonium sulfate precipitation of the enzyme resulted in an increase in specific activity. At between 38% and 45% saturated ammonium sulfate, 20% of the ATPase activity was precipitated, with a specific activity 4 to 5 times higher than that of the crude enzyme. The precipitate was highly sensitive to DCCD.The properties of the ammonium sulfate preparation were investigated. It contained levels of cytochrome and NADH dehydrogenase contamination comparable to those of the highly purified F(0)F(1) preparations from animal tissue. The high degree of purification was corroborated by sodium dodecyl sulfate electrophoresis.

An improved method for the purification of bovine heart mitochondrial transhydrogenase.

An improved procedure for the purification of bovine heart mitochondrial pyridine dinucleotide transhydrogenase is described. The enzyme is purified over 100-fold from submitochondrial particles with a 47.4% yield and a specific activity of 62.3 mumol/min/mg. Heart submitochondrial particle membranes were washed with 2 M NaCl to remove peripheral proteins. This was followed by a 1.5% Triton X-100 extraction of the membranes. The extract was then applied to an affinity column of NAD immobilized to agarose and the enzyme eluted with NADH. This step yielded homogeneous enzyme when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The preparation, when reconstituted into phosphatidylcholine liposomes, coupled proton uptake by the vesicles to the reduction of 3-acetylpyridine adenine dinucleotide by NADPH. The polarity characteristics of transhydrogenase were evaluated by the phase separation technique of Bordier (Bordier, C. (1981) J. Biol. Chem. 256, 1604-1607); 24% of the enzyme partitioned into the aqueous phase and 76% partitioned into the detergent phase. The amino acid composition was determined and polarity index was calculated to be 40%. These data indicate that the enzyme has hydrophilic as well as hydrophobic characteristics.

Kinetics of the potential-sensitive extrinsic probe oxonol VI in beef heart submitochondrial particles

The interaction of the potential-sensitive extrinsic probe oxonol VI with beef heart submitochondrial particles has been investigated under time resolved and equilibrium conditions. The time course of the probe absorption spectrum red shift induced by ATP or NADH injection into a suspension of submitochondrial particles in a dye solution is biphasic, consisting of a faster process described by a second-order rate law with k2 approximately 3 x 10(5) M-1 sec-1. For the ATP pulse experiments, the slower process follows first-order kinetics with k1 approximately 0.3 sec-1. In oxygen pulse experiments to an anaerobic dye-particle system, the slower process is not significantly developed due to rapid depletion of the oxygen, but the faster process follows second-order kinetics with the same rate of the oxygen, but the faster process follows second-order kinetics with the same rate constant as for the ATP and NADH cases. Evidence for permeation of the submitochondrial particle membrane by oxonol VI has been obtained; the slower process is interpretable as describing the permeation of the membrane bilayer. The results of the time-resolved work are consistent with a mechanism involving a redistribution of the dye from the bulk phase to the particle membrane. The value of the second-order rate constant for passive binding of the dye to submitochondrial particles is not compatible with a mechanism proposed to explain the microsecond probe response times in bilayer and excitable membrane experiments nor are such rapid signals observed in the oxonol VI-submitochondrial particle system.

Regulation of succinate dehydrogenase: Does the membrane environment change the catalytic activity of the enzyme?

The factors controlling succinate dehydrogenase activity are discussed. The increase of catalytic activity of the enzyme measured as PMS reduction after binding of reconstitutively active succinate dehydrogenase to the membrane of submitochondrial particles is observed. The hypothesis about the effect of the specific membrane environment on succinate dehydrogenase activity is critically evaluated. It is shown that the effect of “allotopic” increase of dehydrogenase activity depends on the artificial electron acceptor used for the turnover number measurement. Free radical WB reveals about two times higher activity of soluble enzyme compared to PMS, providing that V max values are obtained by extrapolation of both electron acceptors to the infinite concentrations. No increase of activity of the enzyme is found after its binding to the membrane, when WB instead of PMS was used as the artificial electron acceptor. It is concluded that: 1) soluble reconstitutively active succinate dehydrogenase has the same turnover number as particulate enzyme does and 2) PMS which is widely used for succinate dehydrogenase assay is not able to reveal the full catalytic capacity of the soluble enzyme.

Catalytic activity of cytochromes c and c1 in mitochondria and submitochondrial particles

1. Beef heart mitochondria have a cytochrome c 1 : c : aa 3 ratio of 0.65 : 1.0 : 1.0 as isolated; Keilin-Hartree submitochondrial particles have a ratio of 0.65 : 0.4 : 1.0. More than 50% of the submitochondrial particle membrane is in the ‘inverted’ configuration, shielding the catalytically active cytochrome c. The ‘endogenous’ cytochrome c of particles turns over at a maximal rate between 450 and 550 s −1 during the oxidation of succinate or ascorbate plus TMPD; the maximal turnover rate for cytochrome c in mitochondria is 300–400 s −1, at 28° – 30°C, pH 7.4. 2. Ascorbate plus N, N, N′, N′-tetramethyl- p-phenylene diamine added to antimycin-treated particles induces anomalous absorption increases between 555 and 565 nm during the aerobic steady state, which disappear upon anaerobiosis; succinate addition abolishes this cycle and permits the partial resolution of cytochrome c 1 and cytochrome c steady states at 552.5–547 nm and 550–556.5 nm, respectively. 3. Cytochrome c 1 is rather more reduced than cytochrome c during the oxidation of succinate and of ascorbate+ N, N, N′, N′-tetramethyl- p-phenylene diamine in both mitochondria and submitochondrial particles; a near equilibrium condition exists between cytochromes c 1 and c in the aerobic steady state, with a rate constant for the c 1 → c reduction step greater than 10 3 s −1. 4. The greater apparent response of the c aa 3 electron transfer step to salts, the hyperbolic inhibition of succinate oxidation by azide and cyanide, and the kinetic behaviour of the succinate-cytochrome c reductase system, are all explicable in terms of a near-equilibrium condition prevailing at the c 1 c step. Endogenous cytochrome c of mitochondria and submitochondrial particles is apparently largely bound to cytochrome aa 3 units in situ. Cytochrome c 1 can either reduce the cytochrome c-cytochrome aa 3 complex directly, or requires only a small extra amount of cytochrome c to carry the full electron transfer flux.

A study of spin-probe solubility in mitochondrial membranes correlated with ATP-dependent conformational changes

Addition of ATP to submitochondrial particles causes considerable changes in the ESR spectra of hydrophobic spin-probe bound to particles which indicate an improvement in probe solubilization in the submitochondrial particle membranes due to energization. This effect is abolished by 2,4-dinitrophenol (10 -4 M) and oligomycin (I μg/mg of protein). At lower concentrations (0.1–0.2 μg/mg), oligomycin, on the contrary, promotes the action of ATP on the submitochondrial particle-bound spinprobe ESR spectra as well as activates the ATP-dependent transhydrogenase reaction in submitochondrial particles.

Fluorochrome Interaction with the Mitochondrial Membrane: THE EFFECT OF ENERGY CONSERVATION

Abstract The interaction of 1-anilino-8-naphthalenesulfonate and 6-p-toluidino-2-naphthalenesulfonate with the membrane of both mitochondria and submitochondrial particles was studied. A fluorescence increase of the added fluorochromes was observed when energy was supplied to the membrane of submitochondrial particles, while a fluorescence decrease was observed with intact mitochondria. Binding studies and a comparison of the two fluorochromes have indicated that polarity changes of the membrane are not measured by 1-anilino-8-naphthalenesulfonate and 6-p-toluidino-2-naphthalenesulfonate. The fluorescence changes associated with energy conservation can be attributed to changes in the binding of the fluorochromes. Either a conformational change or a membrane potential change could account for the results.

Conversion of biomembrane-produced energy into electric form. III. Chromatophores of Rhodospirillum rubrum

The mechanisms of energy coupling and ion transport in Rhodospirillum rubrum chromatophores have been studied. Photoreduction of NAD + and photophosphorylation have been measured under anaerobic conditions in the presence of N,N,N′,N′-tetramethyl- p-phenylenediamine (TMPD), ascorbate and antimycin A. The uncoupler p-trifluoromethoxycarbonyl cyanide phenylhydrazone (FCCP), as well as ADP + phosphate, has been found to inhibit the photoreduction of NAD + in this system. Addition of an electron acceptor, such as methylviologen, fumarate or O 2, to antimycin-treated chromatophores initiates the process of photophosphorylation. Chromatophores of R. rubrum accumulate the penetrating anions, phenyl dicarboundecaborane and tetraphenyl boron, as well as iodide, if the I − carrier, di-(pentafluorophenyl) mercury, is added. The anion accumulation can be supported by light-induced cyclic electron flow (NADH → O 2, succinate → ferricyanide), by hydrolysis of ATP or inorganic pyrophosphate, as well as by reversal of the energy-requiring transhydrogenase reaction (NADPH → NAD +). The type of energy source influences only the extent of the anion accumulation process. Cessation of the energy supply ( e.g. by exhaustion of the energy source or poisoning of the system by specific inhibitors or an uncoupler) brings about an efflux of the accumulated anions. Uptake of anions is accompanied by alkalinization of the outer solution; release of anions is accompanied by acidification. It is concluded that there is an energy-dependent charge-specific mechanism for anion accumulation in the chromatophore membrane resembling that found in the membrane of submitochondrial particles. It is stated that the electric field (the “ plus” inside the chromatophore) is the motive force for ion transfer through the chromatophore membrane against a concentration gradient. The data on NAD + photoreduction, noncyclic photophosphorylation and energy-dependent anion transport are summarized as the concept of four sites of energy coupling in the chromatophore redox chain localized at the same steps as in animal mitochondria (NADPH → NAD +, NADH → cytochrome b, cytochrome b → cytochrome c, and the region after cytochrome c). Each of these coupling sites can provide energy for generation of a membrane potential.