Proton transport by bacteriorhodopsin through an interface film

Summary Interface films of purple membrane and lipid containing spectroscopically intact and oriented bacteriorhodopsin have been used as a model system to study the function of this protein. Small positive charges in surface potential (<1 mV) are detected upon illumination of these films at the air-water interface. These photopotentials, are not affected by overlaying the interface film with a thin layer (0.3 mm) of decane. However, they are dramatically increased when lipid soluble proton carriers FCCP or DNP are added to the decane. The polarity of the photopotential indicates that, in the light, positive charges are transported through the interface from the aqueous to the organic phase. The action spectrum of the photopotential is identical to the absorption spectrum of bacteriorhodopsin. Since bacteriorhodopsin molecules are oriented with their intracellular surface towards the aqueous subphase, the characteristics of the photopotential indicate that in the light bacteriorhodopsin translocates protons from its intracellular to its extracellular surface. The kinetics of the photopotential reveal that the rate and extent of proton transport are proportional both to the fraction of bacteriorhodopsin molecules excited and to the concentration of proton acceptor. The photopotentials result from changes in the ionic distribution across the decane-water interface and can be cancelled by lipid soluble anions.     

Proton conductance caused by long-chain fatty acids in phospholipid bilayer membranes

Summary Mechanisms of proton conductance (G _H) were investigated in phospholipid bilayer membranes containing long-chain fatty acids (lauric, myristic, palmitic, oleic or phytanic). Membranes were formed from diphytanoyl phosphatidylcholine in decane plus chlorodecane (usually 30% vol/vol). Fatty acids were added either to the aqueous phase or to the membrane-forming solution. Proton conductance was calculated from the steadystate total conductance and the H⁺ diffusion potential produced by a transmembrane pH gradient. Fatty acids causedG _H to increase in proportion to the first power of the fatty acid concentration. TheG _H induced by fatty acids was inhibited by phloretin, low pH and serum albumin.G _H was increased by chlorodecane, and the voltage dependence ofG _H was superlinear. The results suggest that fatty acids act as simple (A^– type) proton carriers. The membrane: water partition coefficient (K _p ) and adsorption coefficient () were estimated by finding the membrane and aqueous fatty acid concentrations which gave identical values ofG _H. For palmitic and oleic acidsK _p was about 10⁵ and was about 10^–2 cm. The A^– translocation or flip-flop rate (k _a ) was estimated from the value ofG _H and the fatty acid concentration in the membrane, assuming that A^– translocation was the rate limiting step in H⁺ transport. Thek _A 's were about 10^–4 sec^–1, slower than classical weak-acid uncouplers by a factor of 10⁵. Although long-chain fatty acids are relatively inefficient H⁺ carriers, they may cause significant biological H⁺ conductance when present in the membrane at high concentrations, e.g., in ischemia, hypoxia, hormonally induced lipolysis, or certain hereditary disorders, e.g., Refsum's (phytanic acid storage) disease.     

Proton flux induced by free fatty acids across phospholipid bilayers: New evidences based on short-circuit measurements in planar lipid membranes

Free fatty acids (FFA) are important mediators of proton transport across membranes. However, information concerning the influence of the structural features of both FFA and the membrane environment on the proton translocation mechanisms across phospholipid membranes is relatively scant. The effects of FFA chain length, unsaturation and membrane composition on proton transport have been addressed in this study by means of electrical measurements in planar lipid bilayers. Proton conductance () was calculated from open-circuit voltage and short-circuit current density measurements. We found that cis-unsaturated FFA caused a more pronounced effect on proton transport as compared to saturated and trans-unsaturated FFA. Cholesterol and cardiolipin decreased membrane leak conductance. Cardiolipin also decreased proton conductance. These effects indicate a dual modulation of protein-independent proton transport by FFA: through a flip-flop mechanism and by modifying a proton diffusional pathway. Moreover the membrane phospholipid composition was shown to importantly affect both processes.     

Proton transport by bacteriorhodopsin in planar membranes assembled from air-water interface films

Bacteriorhodopsin, in known amounts and controlled orientation, is incorporated into planar membrane films. These films are formed by the sequential transfer of two air-water interface films onto a thin, hydrophilic, electrically conductive support cast from nitrocellulose. The films are easily accessible to electrical measurements and to control of the ionic milieu on either side of the membrane. The area of the assembled membrane films can be varied between 2.3 x 10(-2) cm2 and 0.7 cm2. Illumination of these films produces photocurrents, photovoltages, and changes in the pH of the surrounding medium. The peak amplitude of the photocurrent increases linearly with light intensity for dim lights, and it approaches a saturating value for brighter lights. In the linear range, the stoichiometry of transport is 0.65 +/- 0.06 protons/absorbed photon. The rate of transport is linearly proportional to light at all intensities tested. The amplitude and kinetics of the photovoltage measured are accurately predicted by the photocurrent generated and the passive electrical features of the film. Parallel measurements of pH and photocurrent reveal that the light-induced changes in pH are fully accounted for by the rate and amount of charge transport across the membrane. Preceding the transport of protons, a transient photovoltage is detected that exhibits no detectable latency, reaches peak in about 80 microseconds, and probably arises from light-induced intramolecular charge displacements.     

A comparison study on ribonuclease A modifications induced by substituted p-benzoquinones

In this paper, we present our investigation on ribonuclease A (RNase) modifications induced by 1,4-benzoquinone (PBQ), 2-methyl-1,4-benzoquinone (MBQ), and 2-chloro-1,4-benzoquinone (CBQ). The goal of the study was to evaluate quinone-induced protein modifications as well as substituent effects, utilizing several techniques such as SDS–PAGE, fluorescence spectroscopy, microscopy, and LC-ESI⁺-QTOF-MS. SDS–PAGE experiments revealed that all quinones modify RNase through oligomerization as well as polymeric aggregation; with CBQ functioning as the most efficient quinone while MBQ was least efficient. The fluorescence emission was found to be less intense and the anisotropy values were found to be slightly higher for the modified RNase compared to the unmodified RNase. UV–Vis spectroscopy indicated that all three quinones formed adducts in which they were covalently linked to RNase. Confocal imaging analysis showed that the presence of CBQ resulted in massive RNase aggregation, while PBQ-treated RNase formed much smaller aggregates. MBQ-treated RNase exhibited micrographic features that closely resembled those of the unmodified RNase. LC-ESI⁺-QTOF-MS studies indicated the nature of PBQ- and CBQ-induced RNase modifications are complex mainly due to simultaneously occurrence of both adduct formation and oligomerization. Kinetic studies on quinone reactivity toward lysine revealed the rank order of CBQ > PBQ ≫ MBQ, based on the second-order rate constants. We also utilized scanning electron microscopy in order to investigate the effect of modified RNase on the biomineralization of salts.     

Reconstitution of sulfobromophthalein transport in erythrocyte membranes induced by bilitranslocase

Bilitranslocase, the protein responsible for the anion translocation at the sinusoidal plasma membrane level in liver, was shown to be able to reconstitute the transport of sulfobromophthalein in liposomes in the past. The protein preparation used in those experiments consisted of two subunits of 35.5 and 37 kDa. The isolated 37 kDa protein, when inserted in erythrocyte membrane vesicles, confers to the particles the ability to carry out an electrogenic transport of sulfobromophthalein. The effect is specific and can be inhibited by monospecific polyclonal antibodies raised against the protein. In may be concluded that the 37 kDa protein band, present in previous preparations of bilitranslocase, is not only a necessary but also a sufficient component of the transport system for bilirubin and functional analogues.     

A model of non-electrogenic cation transport through bilayer lipid membranes

Quantitative relationship between the proton diffusion potential in the unstirred layers near BLM and NH4Cl was investigated. It has been found that in the range of low concentrations of NH4Cl the potential value depends on the difference of salt concentrations on different sides of the membrane. At higher concentrations the potential value is the function of the ratio of salt concentrations at different BLM sides. In the limiting case the potential value equals 58 mV with NH4Cl concentrations ratio equaling ten. A model is suggested which quantitatively describes the experimental data. It has been shown that the results obtained can be used in determining BLM permeability for weak acids and bases.     

Proton extrusion and attendant transport phenomena in Candida utilis induced by ethanol

Summary Ion fluxes after ethanol addition to Candida utilis depend crucially on aeration (air versus oxygen). In O₂-aerated non-growing cells ethanol causes an H ⁺ / K ⁺ exchange and an extrusion of acetate and lactate accompanied mostly by K ⁺, and their subsequent reimportation together with H ⁺. Cells from continuous culture display generally stronger acidification and more marked K ⁺ movements than non-growing ones. Offprint requests to: A. Prell     

Proton transport through influenza A virus M2 protein reconstituted in vesicles

Influenza A virus M2 protein is known to form acid-activated, proton-selective, amantadine-sensitive channels. We directly measured proton uptake in vesicles containing reconstituted M2 by monitoring external pH after addition of valinomycin to vesicles with 100-fold-diluted external [K⁺]. External pH typically increased by a few tenths of a pH unit over a few minutes after valinomycin addition, but proton uptake was not significantly altered by acidification. Under neutral conditions, external addition of 1 mM amantadine produced a reduction in flux consistent with randomly ordered channels; however, experimental variation is high with this method and the block was not statistically significant. Amantadine block was reduced at pH 5.4. In accord with Lin and Schroeder's study of reconstituted M2 using a pH-sensitive dye to monitor intravesicular pH, we conclude that bath pH weakly affects or does not significantly affect proton flow in the pH range 5.4-7.0 for the reconstituted system, contrary to results from electrophysiological studies. Theoretical analysis of the relaxation to Donnan equilibrium utilized for such vesicle uptake assays illuminates the appropriate timescale of the initial slope and an important limitation that must be placed on inferences about channel ion selectivity. The rise in pH over 10 s after ionophore addition yielded time-averaged single-channel conductances of 0.35 ± 0.20 aS and 0.72 ± 0.42 aS at pH 5.4 and 7.0, respectively, an order of magnitude lower than previously reported in vesicles. Assuming complete membrane incorporation and tetramerization of the reconstituted protein, such a low time-averaged conductance in the face of previously observed single-channel conductance (6 pS at pH 3) implies an open channel probability of 10⁻⁶-10⁻⁴. Based on leakage of potassium from M2-containing vesicles, compared to protein-free vesicles, we conclude that M2 exhibits ∼10⁷ selectivity for hydrogen over potassium.     

A theoretical model for electron transport through chlorophyll-containing bileaflet membranes

Summary A model of electron transport through bileaflet membranes comprised of chlorophylls and carotenoids is proposed and can account for the photoresponses observed in chloroplast-extract membranes. Some features of the model are as follows: In the absence of any specific additive, such as phycocyanin, the energy barrier for electron transfer between chromophores in the membrane and a redox pair in the aqueous solution is significantly high, so that the only realistic mechanism for electron exchange through the interface is by quantum-mechanical tunneling. In an analogy to theories developed for the explanation of semiconductor electrode behavior, the essential requirement of energy overlap between occupied and unoccupied electronic states on either side of the membrane-water interface is explored. The extent of overlap can be controlled by the potential difference developed across the interface. The presence of the carotenoids can reduce the energy barrier for electron movement within she membrane so that excited electrons can pass from the chlorophyll chromophores on one tide of the membrane interface to those on the other side. It is suggested that electron tunneling is the mechanism by which reducing substances belonging to a redox pair of high oxidation-reduction potential deliver their electrons to vacant orbitals in the photosynthetic membranes.     

Proton transport behavior through the influenza A M2 channel: insights from molecular simulation

The structural properties of the influenza A virus M2 transmembrane channel in dimyristoylphosphatidylcholine bilayer for each of the four protonation states of the proton-gating His-37 tetrad and their effects on proton transport for this low-pH activated, highly proton-selective channel are studied by classical molecular dynamics with the multistate empirical valence-bond (MS-EVB) methodology. The excess proton permeation free energy profile and maximum ion conductance calculated from the MS-EVB simulation data combined with the Poisson-Nernst-Planck theory indicates that the triply protonated His-37 state is the most likely open state via a significant side-chain conformational change of the His-37 tetrad. This proposed open state of M2 has a calculated proton permeation free energy barrier of 7 kcal/mol and a maximum conductance of 53 pS compared to the experimental value of 6 pS. By contrast, the maximum conductance for Na(+) is calculated to be four orders of magnitude lower, in reasonable agreement with the experimentally observed proton selectivity. The pH value to activate the channel opening is estimated to be 5.5 from dielectric continuum theory, which is also consistent with experimental results. This study further reveals that the Ala-29 residue region is the primary binding site for the antiflu drug amantadine (AMT), probably because that domain is relatively spacious and hydrophobic. The presence of AMT is calculated to reduce the proton conductance by 99.8% due to a significant dehydration penalty of the excess proton in the vicinity of the channel-bound AMT.     

Ion transport by lasalocid A across red-blood-cell membranes. A multinuclear NMR study

Na+ and K+ fluxes mediated by lasalocid A across erythrocyte membranes have been determined from 23Na-NMR peak areas and chemical shifts, respectively. In similar experiments, Cl- transport has been monitored by NMR signal intensities. Taking into account the external pH variations, the results are readily explainable in terms of charge-balance conservation. The effect of disodium 4,4'-diisothiocyanostilbene-2,2'-disulfonate, an anion-exchange inhibitor, has also been studied.     

Proton/hydroxide conductance through phospholipid bilayer membranes: effects of phytanic acid

Mechanisms of proton/hydroxide conductance (GH/OH) were investigated in planar (Mueller-Rudin) bilayer membranes made from decane solutions of phospholipids or phospholipids plus phytanic acid (a 20-carbon, branched chain fatty acid). At neutral pH, membranes made from diphytanoylphosphatidylcholine or bacterial phosphatidylethanolamine had GH/OH values in the range of (2-5) X 10(-9) S X cm-2, corresponding to H+/OH- 'net' permeabilities of about (0.4-1.0) X 10(-5) cm X s-1. GH/OH was inhibited by serum albumin, phloretin, glycerol and low pH, but was increased by chlorodecane and voltage greater than 80 mV. Water permeability and GH/OH were not correlated, suggesting that water and H+/OH- cross the membrane by separate pathways. Addition of phytanic acid to the phospholipids caused an increase in GH/OH which was proportional to the first power of the phytanic acid concentration. In membranes containing phytanic acid, GH/OH was inhibited by albumin, phloretin, glycerol and low pH, but was increased by chlorodecane and voltages greater than 80 mV. The results suggest that phytanic acid acts as a simple (A- type) proton carrier. The qualitative similarities between the behavior of GH/OH in unmodified and phytanic-acid containing membranes suggest that phospholipids may contain weakly acidic contaminants which cause most of GH/OH at pH greater than 4. However, there is also a significant background (pH independent) GH/OH which may be due to hydrogen-bonded water chains. The ability of phytanic acid to act as a proton carrier may help to explain the toxicity of phytanic acid in Refsum's disease, a metabolic disorder in which phytanic acid accumulates to high levels in plasma, cells and tissues.     

Growth inhibition of Clostridium thermocellum by carboxylic acids: A mechanism based on uncoupling by weak acids

Summary The inhibition of Clostridium thermocellum strains by acetate and other organic acids (propionate, butyrate) can be explained by a model based on the chemiosmotic theory and uncoupler action. It is proposed that the charged permeant species in the process of anion exclusion is the dimer HA _- ² . Evidence for this mechanisms is provided by ³¹P-NMR studies of whole cells and cell extracts.Abbreviations CM4-Cb CM4-cellobiose - EPC⁵⁰ equipotency concentration - P _m bilayer partition coefficient - ka acid dissociation constant     

Proline accumulation induced by weak acids and IAA in coleoptiles of wheat seedlings

Proline accumulation in coleoptiles of wheat seedlings or in excised coleoptile segments incubated under shaking for a 24 h period was studied. There was no increase of proline content of coleoptiles after incubation of the seedlings in 5 mM citric acid (a relatively strong and slowly penetrating organic acid) in a pH range from 4.5 to 7.0 and only a slight increase of proline content after incubation in phosphate buffer at pH 7.0 to 7.5 duo to the higher osmotic concentration of phosphate buffer in this pH range. Quite different results were obtained with seedlings incubated in 10 mM acetic acid, a weak and easily penetrating organic acid. With increasing proton concentrations, proline accumulation increased. Application of 400 mM mannitol or higher concentrations of IAA (more than 10⁻⁵M) additionally increased proline accumulation in the presence of 10 mM acetic acid in the pH range from 6.0 to 7.5 in which acetic acid alone was loss effective. It is suggested that a decrease of cytosolic pH causes stress—induced proline accumulation.