Does the energy state of mitochondria influence the surface potential of the inner mitochondrial membrane? A critical appraisal

Binding of 8-anilino-1-naphthalene sulphonate (ANS) to rat liver mitochondria and submitochondrial inside-out particles was measured under energized and de-energized conditions. In mitochondria, energization/de-energization changed the binding capacity for ANS extrapolated for its infinitely high concentration, whereas the apparent Kd value remained unchanged. In submitochondrial particles apparent Kd was changed but the extrapolated maximum binding was not altered. These results are compatible with theoretical considerations assuming a free permeability of mitochondrial membranes to ANS and its distribution according to the transmembrane potential. The spin-labelled cationic amphiphile, 4-(dodecyl dimethyl ammonium)-1-oxyl-2,2,6,6-tetramethyl piperidine bromide (CAT12), was trapped by de-energized mitochondria in such a way that about half of the bound probe became inaccessible to reduction by externally added ascorbate. This inaccessible fraction was increased by energization. This indicates that this cationic probe can penetrate through the inner mitochondrial membrane. De-energization produced a parallel shift of the Lineweaver-Burk plots for the oxidation of external ferrocytochrome c by mitoplasts and of succinate by submitochondrial particles. A similar shift was obtained by a partial inhibition of succinate oxidation by antimycin A. Thus, the observed changes of the kinetics of the two membrane-bound enzyme systems on de-energization can be interpreted as reflecting changes of the control points of mitochondrial respiration rather than changes of the surface potential. It is concluded that neither the fluorescent probe ANS, the spin-labelled amphiphilic cation CAT12, nor the kinetics of some respiratory enzyme systems provide a sufficient proof for changes of the surface potential of the inner mitochondrial membrane upon energization.     

DCCD inhibits proton translocation and electron flow at the second site of the mitochondrial respiratory chain

DCCD inhibits formation of a succinate-driven transmembrane pH gradient in submitochondrial particles, as shown by inhibition of fluorescence quenching of 9-aminoacridine, without concomitant inhibition of succinate oxidation. On the other hand ubiquinol-cytochrome c reductase activity is inhibited by DCCD. Half-inhibition of both fluorescence quenching and ubiquinol-cytochrome c reductase occur at 35 μM DCCD. The results suggest that DCCD inhibits proton pumping activity coupled to electron flow through the bc₁ complex.     

Fluorescence study of lymphocyte plasma membranes]

ANS binding parameters--dissociation constant, number of binding sites, rotation freedom--are measured by fluorescence studies of a complex between ANS and lymph node cell plasma membranes. Divalent ions, Mg++ and Ca++, enhance the complex fluorescence intensity without shifting its maximum wavelength : this enhancement is induced by affinity and quantum yield increases, while the number of binding sites remains constant. The complex fluorescence quenching by ethacrynic acid shows the presence of free SH groups in the ANS binding site. An energy transfer takes place between membrane protein tryptophan residues and bound ANS ; the energy transfer yield is unaffected by Ca++ ions. A correlation of these results is postulated with the biological activity of the membrane.     

Quantitation of the energetic state in mitochondria and submitochondrial vesicles with 8-anilino-1-naphthalene sulfonic acid

Some of the apparently anomalous findings made with the fluorescent probe 8-anilino-1-naphthalene sulfonic acid (ANS) have been reinvestigated using rat liver mitochondria. The results have been found compatible with current views on energy conservation.The direction of fluorescence and proton flux changes under different conditions have been delineated. The relation of these results to consideration of membrane polarity and organization is discussed.The reliability of ANS fluorescence changes in determining the level of energization of mitochondria and submitochondrial preparations is discussed.Abbreviations used ANS 8-anilino-1-naphthalene sulfonic acid - F _E and H⁺ _E O₂ dependent change in fluorescence and H⁺ in mitochondria and SMP - SMP submitochondrial preparation     

Use of fluorescence probes 1-aniline-8-naphthalene sulfonate and pyrene for studying the localisation of proteins in inner membranes from wheat etioplasts

The fluorescence probes 1-aniline-8-naphthalene sulfonate (ANS) and pyrene were applied for characterisation of the light-induced changes in etioplast inner membranes (EPIMs) from 7 d-old dark-grown wheat seedlings (Triticum aestivum L. cv. Pobeda). The major aim was to obtain information about the localisation of membrane proteins in the EPIMs, using probes situated in different regions of the membranes. The quenching of tryptophan fluorescence showed tha the main parts of proteins were accessible to the pyrene buried in the lipid bilayer which suggests that most of the proteins also enter the lipid bilayer. The substantial quenching of the tryptophan fluorescence by the surface-situated ANS demonstrated that a part of the tryptophan residues was probably localised close to the membrane surface. The registered changes after irradiation could be explained by the presence of large aggregates of NADPH-protochlorophyllide oxidoreductase (POR), protochlorophyllide (PChlide) and NADPH in membranes that start to disconnect and redistribute along the prothylakoids. This revised version was published online in August 2006 with corrections to the Cover Date.     

Energy transfer from tryptophan residues of proteins to 8-anilinonaphthalene-1-sulfonate

The binding of the apolar fluorescent dye 8-anilinonaphthalene-1-sulfonate (ANS) to bovine serum albumin (BSA), phospholipase A₂ (PLA₂), ovalbumin, lysozyme, cobrotoxin and N-acetyltryptophanamide was used to assess the factors affecting the efficiency of energy transfer from Trp residues to the ANS molecule. We found that the efficiency of energy transfer from Trp residues to ANS was associated with the ability of proteins to enhance the ANS fluorescence. At the same molar concentration of protein, BSA enhanced ANS fluorescence most among these proteins; its Trp fluorescence was drastically quenched by the addition of ANS. Fluorescence enhancement of ANS in PLA₂-ANS complex increased upon addition of Ca²⁺ or change of the buffer to acidicpH, resulting in a higher efficiency of energy transfer from Trp residues to ANS. There was limited ANS fluorescence enhancement with ovalbumin, lysozyme, cobrotoxin, and N-acetyltryptophanamide and a less efficient quenching in Trp fluorescence. The capabilities of proteins for binding with ANS correlated with the decrease in their Trp fluorescence being quenching by ANS. However, the microenvironment surrounding Trp residues of proteins did not affect the energy transfer. Based on these results, the factors that affected the energy transfer from Trp residues to ANS are discussed.     

An analysis of the binding of fluorescence probes in mitochondrial systems.

Measurements of the binding of the fluorescent probes 8-anilinonaphthalene-1-sulfonate (ANS) and ethidium ions to whole and disruped mitochondria and submitochondrial particles suggest that the inner mitochondrial membrane is freely permeable to the two probes. Equations relating the binding of permeant probes to the electro-chemical balance across the membrane of vesicular systems are derived and these equations used to analyze Scatchard plots of the binding of the two probes to energized and nonenergized mitochondria and EDTA particles.     

Partial characterization of the 1-anilino-8-naphthalene sulfonate-adrenochrome semicarbazide interaction site in erythrocyte ghost membrane fragments.

The effect of adrenochrome semicarbazide on the conformation of erythrocyte ghost membranes has been studied by ANS fluorescence, lipid and sulfhydryl spin labels and circular dichroism. No large conformational alterations in the membrane were detected by these techniques. Noncompetitive quenching of ANS fluorescence by ADCS suggests ADCS to interact with the membrane at sites close to the ANS binding domain.     

An analysis of the binding of fluorescence probes in mitochondrial systems

Summary Measurements of the binding of the fluorescent probes 8-anilinonaphthalene-1-sulfonate (ANS) and ethidium ions to whole and disrupted mitochondria and submitochondrial particles suggest that the inner mitochondrial membrane is freely permeable to the two probes. Equations relating the binding of permeant probes to the electro-chemical balance across the membrane of vesicular systems are derived and these equations used to analyze Scatchard plots of the binding of the two probes to energized and nonenergized mitochondria and EDTA particles.     

Mechanism of inhibition of Ca2+-transport activity of sarcoplasmic reticulum Ca2+-ATPase by anisodamine

The mechanism of inhibition of Ca2+-transport activity of rabbit sarcoplasmic reticulum Ca 2+-ATPase (SERCA) by anisodamine (a drug isolated from a medicinal herb Hyoscyamuns niger L) was investigated by using ANS (1-anilino-8-naphthalenesulfonate) fluorescence probe, intrinsic fluorescence quenching and Ca 2+-transport activity assays. The number of ANS binding sites for apo Ca2+-ATPase was determined as 8, using a multiple-identical binding site model. Both anisodamine and Ca2+ at millimolar level enhanced the ANS binding fluorescence intensities. Only anisodamine increased the number of ANS molecules bound by SERCA from 8 to 14. The dissociation constants of ANS to the enzyme without any ligand, with 30 mM anisodamine and with 15 mM Ca 2 were found to be 53.0 microM, 85.0 microM and 50.1 microM, respectively. Both anisodamine and Ca2+ enhanced the ANS binding fluorescenc with apparent dissociation constants of 7.6 mM and 2.3 mM, respectively, at a constant concentration of the enzyme. Binding of anisodamine significantly decreased the binding capacity of Ca2+ with the dissociation constant of 9.5 mM, but binding of Ca2+ had no obvious effect on binding of anisodamine. Intrinsic fluorescence quenching and Ca2+-transport activity assays gave the dissociation constants of anisodamine to SERCA as 9.7 and 5.4 mM, respectively, which were consistent with those obtained from ANS-binding fluorescence changes during titration of SERCA with anisodamine and anisodamine + 15 mM Ca2+, respectively. The results suggest that anisodamine regulates Ca2+-transport activity of the enzyme, by stabilizing the trans-membrane domain in an expanded, inactive conformation, at least at its annular ring region.     

Some properties of mitochondria, mitoplasts and submitochondrial particles of different polarities from plant tissues

Irrespective of the starting material, i.e. washed mitochondria, purified mitochondria or mitoplast from Solanum tuberosum L., submitochondrial particles of well-defined polarities can be generated by French press treatment in low-salt medium or by sonication in high-salt medium. The first treatment will result in submitochondrial particles which are more than 80% right-side-out (right-side-out particles), the second in submitochondrial particles that are more than 80% inside-out (inside-out particles). The isoelectric point (pI = 4.0) of the inside-out particles measured by cross-partition is distinctly different from the isoelectric points of the other mitochondrial membranes which exhibit pI values between 4.5 and 4.7. The surface charge density measured by 9-aminoacridine fluorescence varies in the same order from −27 mC · m⁻² for Percoll-purified mitochondria to −51 mC · m⁻² for both right-side-out and inside-out particles. Even though the charge densities for the two surfaces of the inner membrane are similar, inside-out particles are much more negatively charged at pH 7.0, since they are 6-times larger. These results clearly demonstrate that it is possible to obtain submitochondrial particles of various polarities and sizes which in turn constitute valuable tools for the study of lateral and transverse asymmetry of the inner mitochondrial membrane.     

Detection of conformational changes in chloroplast coupling factor 1 by 8-anilino-1-naphthalene-sulphonate fluorescence changes

Chloroplast coupling factor 1 (CF1) contains a high-affinity binding site for 8-anilino-1-napthalene sulphonate (ANS,Kd = 5-6 microM). The binding of ANS to the enzyme is associated with a fluorescence enhancement and a blue-shift in the emission spectrum. ANS only slightly inhibits ATP hydrolysis by CF1. Adenine nucleotides and inorganic phosphate induce a fast ANS fluorescence quenching of about 50% which is due to a decrease in the affinity of the enzyme for ANS (Kd increases from 6 microM to 22 microM) and in the fluorescence quantum yield of the bound probe (by 33%) but not in the number of ANS sites (n = 1). Conversely, Mg and Ca ions induce a fluorescence enhancement of bound ANS. Inactivation of the enzyme enhances ANS fluorescence, eliminates the response to adenine nucleotides and inorganic phosphate but increases the response to divalent metals. The affinity of latent CF1 for ADP (Kd = 12 microM) is considerably higher than for ATP (Kd = 95 microM) in buffer containing EDTA. The Kd for inorganic phosphate is 140 microM. Mg increases the apparent affinity for ATP (Kd = 28 microM) but not for ADP or Pi. Binding of ATP to the tight-sites does not inhibit the ADP or Pi-induced fluorescence quenching but decreases the affinity for ADP (Kd = 34 microM) and for inorganic phosphate (Kd = 320 microM). These results suggest that the ADP and phosphate binding sites are different but not independent from the tight sites. Activation of a Mg-specific ATPase in CF1 by octyl glucoside decreases the affinity for ADP and inorganic phosphate by about threefold but increases the affinity for ATP. ATPase activation of CF1 also increases the Ki for ADP inhibition of ATP hydrolysis. ATPase activation also influences the ANS responses to Ca and Mg. Ca-ATPase activation increases the fluorescence enhancement and the apparent affinity for Ca whereas Mg-ATPase activation specifically increases the Mg-induced fluorescence enhancement. The fluorescence of CF1-bound ANS is enhanced by Dio-9 and quenched by phloridzin, quercetin, Nbf-Cl and FITC. Nbf-Cl and FITC completely inhibit the ADP-induced fluorescence quenching whereas Dio-9 inhibits the Mg-induced fluorescence enhancement. ANS does not relieve the quercetin or phloridzin inhibition of ATP hydrolysis indicating that these inhibitors do not compete with ANS for a common binding site. ANS may be used, therefore, as a sensitive probe to detect conformational changes in CF1 in response to activation or inactivation and to binding of substrates and of inhibitors.     

Binding of [14C] DDT by submitochondrial particles

1. Binding of [14C] DDT by submitochondrial particles and by liposomes prepared from lipids extracted from the particles was studied by the discontinuous sucrose gradient method. 2. Binding of the insecticide was a biphasic linear function of the biomembrane- and liposome-concentration with a break in the binding curve occurring at identical concentrations of phospholipid for both the biomembrane and vesicle. The biphasic binding curve is interpreted in terms of decreased availability of binding sites as a result of particle-particle interaction. 3. [14C] DDT was bound mainly by the membrane lipids and only negligible binding was detected for the delipidated membrane. 4. A 100-200-fold excess of unlabeled DDT had no effect on the binding of [14C] DDT and a 600-fold excess of unlabeled DDT reduced the binding by 20% suggesting that binding of [14C] DDT by lipids was nonspecific. 5. These results are discussed in relation to the strong inhibition by DDT of mitochondrial bioenergetics.     

The structural characteristics of human preprotein translocase of the inner mitochondrial membrane Tim23: implications for its physiological activities

The preprotein translocase of the inner mitochondrial membrane (TIM23 complex) is the main entry gate for proteins of the matrix and the inner membrane. Tim23 forms a pore for preprotein transportation in TIM23 complex, which spans the inner membrane with transmembrane segments and exposes a hydrophilic domain in the intermembrane space. In this study, we expressed and purified the intermembrane space (IMS) domain of human Tim23 (Tim23(IMS)). The far-UV CD spectra of Tim23(IMS) in native and denatured states revealed that the protein has a limited secondary structure and a not well-defined tertiary packing. Its Stokes radius was larger than both its expected size as a folded globular protein and the size determined by size exclusion chromatography. A large increase in 8-anilino-1-naphthalene-sulfonate (ANS) fluorescence (>50-fold) was observed, indicating that hydrophobic clusters are exposed at its surface. And GlobPlot/DisEMBL program predicted that the protein is in a loose folding state. We therefore conclude that, the non-bound hydrophilic domain of the human Tim23 is in a molten globule configuration with marginal stability. Furthermore, size exclusion chromatography and sedimentation equilibrium analysis showed that Tim23(IMS) exists as a dimer. And the results, showed by ANS binding and fluorescence quenching, indicated that a pH-dependent conformational change of Tim23(IMS) occurs, and at pH 4 and 3, it forms a compact structure.     

汞化合物对红细胞膜作用的研究

本文研究了汞化合物对红细胞膜作用的光谱变化,观察了膜蛋白的荧光和磷光,膜上DPH的荧光偏振和ANS与红细胞膜的结合,以及他们与汞产生红血球溶血的关系。 在5P7.5缓冲液中红细胞膜蛋白的荧光随HgCl_2 Hg(AC)_2和PCMB的浓度加大而降低,表现为快和慢双相变化的过程,其淬灭作用的大小为HgCl_2>Hg(AC)_2PCMB,这是由于膜上形成了不发荧光的R—Trp—Hg~ 络合物以及能量从Trp转移到R—S—Hg~ 络合物上。HgCl_2对膜蛋白磷光的作用也是随汞离子浓度加大而降低,但磷光/荧光比则是增加的。标记红细胞膜的DPH偏振度是随HgCl_2浓度增加,表明膜流动性是随汞离子浓度加大而降低。标记膜上的ANS的荧光强度随HgCl_2和Hg(AC)_2的浓度加大而增加,这是由于ANS与膜的结合数随汞离子浓度加大而增加的缘故。上述各种变化是与汞离子对红血球溶血的作用一致的。     

The effect of surface and membrane potential on 1-anilino-8-naphthalenesulfonate binding with E. coli membrane

Dependence of ANS fluorescence on the surface potential of E. coli under lowered resistance of the bacterial membrane and after application of the positive diffusion potential inside the cell was investigated. It was shown that in the absence of the latter ANS binding in de-energised bacteria occurs mainly at the outside surface. It may be due to the high negative charge of the inner side of the cytoplasmic membrane. According to produced evaluation the potential of this surface is 120 +/- 25 mV. The data obtained suggest that low ANS fluorescence in intact cells is due to the membrane modification on energisation.     

The interaction of the anionic fluorescence probe, 1-anilinonaphthalene-8-sulfonate, with hepatocytes and hepatoma tissue culture cells

The fluorescence probe 1-anilinonaphthalene-8-sulfonate (ANS) has been used to characterize the anion transport properties of normal hepatocytes and hepatoma tissue culture cells. Incubation of hepatocytes in the presence of ANS (20 micron) resulted in a 35-fold enhancement of fluorescence and a 50 nm blue shift. The time course of this process is biphasic. A rapid initial fluorescence enhancement suggests ANS binding to the plasma membrane, and a slower component reflects the uptake of ANS into intracellular compartments. Analysis of ANS uptake showed this latter process to be saturable, with a Km of 10 micron, to be temperature dependent and to occur only in viable cells. The above observations suggest a carrier-mediated anion transport mechanism. Incubation of hepatoma tissue culture cells with ANS (20 micron) gave a fluorescence emission spectrum similar to that obtained from purified plasma membranes. The kinetics of this interaction only exhibited a rapid initial binding of ANS. The second slow component was now absent, suggesting that ANS transport by the malignant cell system was greatly reduced. Transport of ANS could, however, be stimulated in the presence of the local anesthetic tetracaine. The observed transport was now saturable, temperature dependent, and as in normal hepatocytes, required viable cells, again indicating a carrier-mediated transport system. These studies suggest a significant alteration in membrane function in hepatoma tissue culture cells resulting in a major defect in anion transport.     

Sidedness of inhibition of energy transduction in oxidative phosphorylation in rat liver mitochondria by ethidium bromide.

Ethidium bromide, a new type of inhibitor of energy transduction in oxidative phosphorylation, inhibited ATP synthesis in intact mitochondria but not in submitochondrial particles, the latter being inside-out relative to the membranes of intact mitochondria. Ethidium bromide incorporated inside the submitochondrial particles inhibited ATP synthesis in the particles. The decrease of the membrane potential by valinomycin (plus KCl) inhibited only slightly the energy-dependent binding of ethidium bromide to the mitochondria. The present results show clearly that ethidium bromide inhibited energy transduction in oxidative phosphorylation by acting on the outer side (C-side) of the inner mitochondrial membrane, perhaps by neutralizing negative charges created on the surface of the C-side, and that it had no inhibitory activity on the inner side (M-side) of the membrane. Th present results show also that the energy-dependent binding of ethidium is not due to electrophoretic transport down the membrane potential; ethidium may bind to negative charges on the surface of the C-side. The present study suggest that an anisotropic distribution of electric charge in the inner mitochondrial membrane is an intermediary high energy state of oxidatvie phosphorylation.     

Attenuation of sugar cataract by ethyl pyruvate

The effects of fluoxetine on the oxidative phosphorylation of mitochondria isolated from rat brain and on the kinetic properties of submitochondrial particle F₁F₀-ATPase were evaluated. The state 3 respiration rate supported by pyruvate + malate, succinate, or ascorbate + tetramethyl-p-phenylenediamine (TMPD) was substantially decreased by fluoxetine. The IC₅₀ for pyruvate + malate oxidation was 0.15 mM and the pattern of inhibition was the typical one of the electron-transport inhibitors, in that the drug inhibited both ADP- and carbonyl cyanide m-chlorophenylhydrazone (CCCP)-stimulated respirations and the former inhibition was not released by the uncoupler. Fluoxetine also decreased the activity of submitochondrial particle F₁F₀-ATPase (IC₅₀ 0.08 mM) even though K_0.5 and activity of Triton X-100 solubilized enzyme were not changed substantially. As a consequence of these effects, fluoxetine decreased the rate of ATP synthesis and depressed the phosphorylation potential of mitochondria. Incubation of mitochondria or submitochondrial particles with fluoxetine under the conditions of respiration or F₁F₀-ATPase assays, respectively, caused a dose-dependent enhancement of 1-anilino-8-naphthalene sulfonate (ANS) fluorescence. These results show that fluoxetine indirectly and nonspecifically affects electron transport and F₁F₀)-ATPase activity inhibiting oxidative phosphorylation in isolated rat brain mitochondria. They suggest, in addition, that these effects are mediated by the drug interference with the physical state of lipid bilayer of inner mitochondrial membrane.     

Interaction of the fluorescent probe, 1-anilino-8-napthalene sulfonate, with the sulfate transport system of Ehrlich ascites tumor cells.

The addition of the fluorescent dye, ANS, to intact ascites tumor cells results in an enhancement of fluorescence intensity. The increase in fluorescence intensity as a function of time is biphasic which suggests that at least two processes occur. The first associated with the rapid initial rise in fluorescence represents binding to the cell surface while the second or slower phase reflects entrance of ANS into the intracellular phase. The relationship between bound and free ANS in 0.50 mM sulfate medium was used to calculate the apparent dissociation constant of ANS-membrane complex (Kd = 6.53 times 10(-5) M) and the total number of ANS binding sites (4.49 nmoles/mg dry weight). Kinetic analysis of steady state sulfate transport in the presence and absence of ANS suggests that (1) sulfate exchange can be described by Michaelis Menten type kinetics (Km = 2.05 times 10(-3) M), (2) a small fraction of surface associated ANS competitively inhibits sulfate exchange (Ki = 4.28 times 10(-6) M) and (3) the transport system has a higher affinity for ANS than for sulfate. These data are consistent with the hypothesis that inhibition of sulfate exchange is related to the direct, reversible interaction of the negatively charged sulfonate group of ANS with superficial positively charged membrane sites.