The apparent permeability coefficient for proton flux through phosphatidylcholine vesicles is dependent on the direction of flux

A dioleoylphosphatidylcholine unilamellar vesicle model system was used to determine proton permeability. The fluorescence of the pH reporter group, pyranine, trapped within vesicles with a difference in pH across the bilayer, was digitized and analyzed with numerical integration. When H+ flux was initiated by the acidification of the external buffer (acid jump), the apparent H+ permeability was found to be a linear function of the reciprocal of the internal H+ concentration with the slope inversely proportional to the initial size of the H+ gradient. When flux was initiated by the alkalinization of the external buffer (base jump), the apparent permeability coefficient was constant for each external H+ concentration. However, the value of the apparent permeability was linearly dependent on the reciprocal of the external H+. The possibility that carbonates (carbon dioxide, carbonic acid, bicarbonate and carbonate) could be acting as proton carriers was tested by adding millimolar concentrations of bicarbonate to solutions greatly reduced in carbonates. The slopes of the graphs of apparent permeability coefficient vs. reciprocal H+ were linear functions of added bicarbonate concentration for both acid and base jump conditions. These observations were interpreted in terms of a model suggesting that carbonic acid or carbon dioxide together with bicarbonate was an efficient proton carrier across phospholipid bilayers.     

Catalytic mechanism of alpha-class carbonic anhydrases: CO2 hydration and proton transfer

Carbonic anhydrases were first identified in red blood cells and have been thus traditionally addressed in a hematological context. However, recently there has been a shift of research interest to therapeutic areas, notably in solid cancers, relegating the impact of carbonic anhydrase function and pathological dysfunction in blood related physiology to secondary importance. This review addresses this paradigm and emphasizes the potential impact of recent studies on blood related carbonic anhydrase isotype expression and modulation in diverse areas such as physiology and pathology, biosensing, their use as biomarkers, and in the development of synthetic blood. A special emphasis is placed on reviewing new dynamic and quantitative studies that allow for the efficient tracking and quantitation of various carbonic anhydrase isozymes within the blood and more generally within the human body, that give new perspectives on the biochemical and physiological role of blood associated carbonic anhydrase in health and pathology.     

Production of phosphatidylcholine containing conjugated linoleic acid mediated by phospholipase A2

Esterification of lysophosphatidylcholine (LPC) with conjugated linoleic acid (CLA) was carried out using porcine pancreatic phospholipase A₂ (PLA₂). PLA₂ only slightly synthesized phosphatidylcholine containing CLA (CLA-PC) at 2.6% by the addition of water. Addition of formamide in place of water markedly increased the yield of CLA-PC. In addition, synthesis of CLA-PC by PLA₂ was affected by the amount of substrate CLA and PLA₂ in the reaction system. Under optimal reaction conditions using 11 mg LPC, 18 mg CLA, 550 mg glycerol, 50 μL formamide, 3.3 × 10⁴ U PLA₂, and 0.3 μmol CaCl₂ at 37 °C for 6 h, the reaction yield of CLA-PC reached 65 mol%. Furthermore, addition of protein such as albumin and casein suppressed the decrease of CLA-PC yield after 6 h. PLA₂ exhibited the highest activity for the 10t,12c-CLA isomer among four CLA isomers (9c,11t-CLA, 9c,11c-CLA, 9t,11t-CLA and 10t,12c-CLA), whereas that for 9c,11c-CLA was the lowest. These results showed that the present esterification system for LPC and CLA by PLA₂ is effective for producing CLA-PC.     

Mechanism of proton permeation through chloroplast lipid membranes.

Electrical measurements were carried out to investigate the contribution of chloroplast lipids to the passive proton permeability of both the thylakoid and inner-envelope membranes. Permeability coefficient and conductance to protons were measured for solvent-free bilayers made from monogalactosyldiglyceride:digalactosyldiglycerid: sulfoquinovosyldiglyceride:phosphatidylglycerol (2:1:0.5:0.5, w/w) in the presence of a pH gradient of 7.4/8.1. The permeability coefficient for protons in glycolipids was 5.5 +/- 1.1 x 10(-4) cm s-1 (n = 14). To determine whether this high H+ permeability could be explained by the presence of lipid contaminants such as weak acids, we investigated the effects of (a) bovine serum albumin, which can remove some amphiphilic molecules such as free fatty acids, (b) 6-ketocholestanol, which increases the membrane dipole potential, (c) oleic acid, and (d) chlorodecane, which increases the dielectric constant of the lipid bilayer. Our results show that free fatty acids are inefficient protonophores, as compared with carbonylcyanide-m-chlorphenythydrazone, and that the hypothesis of a weak acid mechanism is not valid with glycolipid bilayers. In the presence of deuterium oxide the H+ conductane was reduced significantly, indicating that proton transport through the glycolipid matrix could occur directly by a hydrogen bond process. The passive transport of H+ through the glycolipid matrix is discussed with regard to the activity of the thylakoid ATP synthase and the inner-envelope H(+)-ATPase.     

Free radical mediated x-ray damage of model membranes.

The damaging effects of synchrotron-derived x rays on aqueous phospholipid dispersions have been evaluated. The effect of degree of lipid hydration, phospholipid chemical structure, mesophase identity, aqueous medium composition, and incident flux on the severity and progress of damage was quantified using time-resolved x-ray diffraction and chromatographic analysis of damage products. Electron spin resonance measurements of spin-trapped intermediates generated during irradiation suggest a free radical-mediated process. Surprisingly, radiation damage effects revealed by x-ray diffraction were imperceptible when the lamellar phases were prepared under water-stressed conditions, despite the fact that x-ray-induced chemical breakdown of the lipid occurred regardless of hydration level. Of the fully hydrated lipid systems studied, saturated diacyl-phosphatidylcholines were most sensitive to radiation damage compared to the ester- and ether-linked phosphatidylethanolamines and the ether-linked phosphatidylcholines. The inclusion of buffers or inorganic salts in the dispersing medium had only a minor effect in reducing damage development. A small inverse dose-rate effect was found when the x-ray beam intensity was changed 15-fold. These results contribute to our understanding of the mechanism of radiation damage, to our appreciation of the importance of monitoring both structure and composition when evaluating biomaterials radiation sensitivity, and to the development of strategies for eliminating or reducing the severity of damage due to an increasingly important source of x rays, synchrotron radiation. Because damage is shown to be free radical mediated, these results have an important bearing on age-related accumulation of free radicals in cells and how these might compromise membrane integrity, culminating in cell death.     

Preparation and properties of vesicles enclosed by fatty acid membranes.

Stable preparations of microscopic particles were obtained from long-chain fatty acids by mechanical agitation of evaporated films in presence of buffer solutions. Oleic and linoleic acids were used. Studies of osmotic swelling and shrinking of the particles indicated that they are enclosed by semipermeable membranes. The particles, which were named ufasomes, are also capable of entrapping glucose in spaces inaccessible to enzymes. It was concluded that the ufasomes closely resemble phospholipid liposomes in their structure and properties.     

Permeability of dimyristoyl phosphatidylcholine/dipalmitoyl phosphatidylcholine bilayer membranes with coexisting gel and liquid-crystalline phases.

The passive permeation of glucose and a small zwitterionic molecule, methyl-phosphoethanolamine, across two-component phospholipid bilayers (dimyristoyl phosphatidylcholine (DMPC)/dipalmitoyl phosphatidylcholine (DPPC) mixtures) exhibit a maximum when gel domains and fluid domains coexist. The permeability data of the two-phase bilayers cannot be fitted to single-rate kinetics, but are consistent with a Gaussian distribution of rate constants. In pure DMPC and DPPC as well as in their mixtures, at the temperature of the maximum excess heat capacity, the logarithm of the average permeability rate constants are linearly correlated with the mole fraction of DPPC in the total system. In addition, in the 50:50 mixture, the excess heat capacity values as well as the apparent fractions of interfacial lipid correlate with the logarithm of the excess permeabilities in the two-phase region. These results suggest that small polar molecules can cross the membrane at the interface between gel and fluid domains at a much faster rate than through the homogeneous phases; the acyl chains located at the domain interface experience lateral density fluctuations that are inversely proportional to their average length, and large enough to allow rapid transmembrane diffusion of the solute molecules. The distribution of the permeability rate constants may reflect temporal and spatial fluctuations of the lipid composition at the phase boundaries.     

Temperature-reversible eruptions of vesicles in model membranes studied by NMR.

Deuterium (2H) and phosphorus (31P) nuclear magnetic resonance (NMR) and freeze-fracture electron microscopy were used to study spontaneous vesiculation in model membranes composed of POPC:POPS with or without cholesterol. The NMR spectra indicated the presence of a central isotropic line, the intensity of which is reversibly and linearly dependent upon temperature in the L alpha phase, with no hysteresis when cycling between higher and lower temperatures. Freeze-fracture microscopy showed small, apparently connected vesicles that were only present when the samples were frozen (for freeze-fracture) from an initial temperature of 40-60 degrees C, and absent when the samples are frozen from an initial temperature of 20 degrees C. Analysis of motional narrowing was consistent with the isotropic lines being due to lateral diffusion in (and tumbling of) small vesicles (diameters approximately 50 nm). These results were interpreted in terms of current theories of shape fluctuations in large unilamellar vesicles which predict that small daughter vesicles may spontaneously "erupt" from larger parent vesicles in order to expel the excess area created by thermal expansion of the bilayer surface at constant volume. Assuming that all the increased area due to increasing temperature is associated with the isotropic lines, the NMR results allowed a novel estimate of the coefficient of area expansion alpha A in multilamellar vesicles (MLVs) which is in good agreement with micromechanical measurements upon giant unilamellar vesicles of similar composition. Experiments performed on unilamellar vesicles, which had been placed upon glass beads, confirmed that alpha A determined in this way is unchanged compared with the MLV case. Addition of the highly positively charged (extrinsic) myelin basic protein (MBP) to a POPC:POPS system showed that membrane eruptions of the type described here occur in response to the presence of this protein.     

Hydration of CO2 by carbonic anhydrase: intramolecular proton transfer between Zn2+-bound H2O and histidine 64 in human carbonic anhydrase II

The energy barrier for the intramolecular proton transfer between zinc-bound water and His 64 in the active site of human carbonic anhydrase II (HCA II) has been studied at the partial retention of diatomic differential overlap (PRDDO) level. The most important stabilizing factor for the intramolecular proton transfer is the zinc ion, which lowers the pKa of zinc-bound water and electrostatically repels the proton. The energy barrier of 127.5 kcal/mol for proton transfer between a water dimer is completely removed in the presence of the zinc ion. The zinc ligands, which donate electrons to the zinc ion, raise the barrier slightly to 34 kcal/mol for a 4-coordinated zinc complex including three imidazole ligands from His 94, His 96, and His 119 and to 54 kcal/mol for the 5-coordinated zinc complex including the fifth water ligand. A few model calculations indicate that these energy barriers are expected to be reduced to within experimental range (approximately 10 kcal/mol) when large basis set, correlation energies, and molecular dynamics are considered. The proton-transfer group, which functions as proton receiver in the intramolecular proton transfer, helps to attract the proton; and the partially ordered active site water molecules are important for proton relay function.     

Cytochrome C interaction with cardiolipin/phosphatidylcholine model membranes: effect of cardiolipin protonation

Resonance energy transfer between anthrylvinyl-labeled phosphatidylcholine as a donor and heme moiety of cytochrome c (cyt c) as an acceptor has been employed to explore the protein binding to model membranes, composed of phosphatidylcholine and cardiolipin (CL). The existence of two types of protein-lipid complexes has been hypothesized where either deprotonated or partially protonated CL molecules are responsible for cyt c attachment to bilayer surface. To quantitatively describe cyt c membrane binding, the adsorption model based on scaled particle and double layer theories has been employed, with potential-dependent association constants being treated as a function of acidic phospholipid mole fraction, degree of CL protonation, ionic strength, and surface coverage. Multiple arrays of resonance energy transfer data obtained under conditions of varying pH, ionic strength, CL content, and protein/lipid molar ratio have been analyzed in terms of the model of energy transfer in two-dimensional systems combined with the adsorption model allowing for area exclusion and electrostatic effects. The set of recovered model parameters included effective protein charge, intrinsic association constants, and heme distance from the bilayer midplane for both types of protein-lipid complexes. Upon increasing CL mole fraction from 10 to 20 mol % (the value close to that characteristic of the inner mitochondrial membrane), the binding equilibrium dramatically shifted toward cyt c association with partially protonated CL species. The estimates of heme distance from bilayer center suggest shallow bilayer location of cyt c at physiological pH, whereas at pH below 6.0, the protein tends to insert into membrane core.     

Cytochrome c induces lipid demixing in weakly charged phosphatidylcholine/phosphatidylglycerol model membranes as evidenced by resonance energy transfer

Resonance energy transfer (RET) between anthrylvinyl-labeled phosphatidylcholine (AV-PC) or phosphatidylglycerol (AV-PG) as donors and the heme groups of cytochrome c (cyt c) as acceptors was examined in PC/PG model membranes containing 10, 20 or 40 mol% PG with an emphasis on evaluating lipid demixing caused by this protein. The differences between AV-PC and AV-PG RET profiles observed at PG content 10 mol% were attributed to cyt c ability to produce segregation of acidic lipids into lateral domains. The radius of lipid domains recovered using Monte-Carlo simulation approach was found not to exceed 4 nm pointing to the local character of cyt c-induced lipid demixing. Increase of the membrane PG content to 20 or 40 mol% resulted in domain dissipation as evidenced by the absence of any RET enhancement while recruiting AV-PG instead of AV-PC.     

Characterization of Avt1p as a vacuolar proton/amino acid antiporter in Saccharomyces cerevisiae

Several genes for vacuolar amino acid transport were reported in Saccharomyces cerevisiae, but have not well been investigated. We characterized AVT1, a member of the AVT vacuolar transporter family, which is reported to be involved in lifespan of yeast. ATP-dependent uptake of isoleucine and histidine by the vacuolar vesicles of an AVT exporter mutant was lost by introducing avt1? mutation. Uptake activity was inhibited by the V-ATPase inhibitor: concanamycin A and a protonophore. Isoleucine uptake was inhibited by various neutral amino acids and histidine, but not by γ-aminobutyric acid, glutamate, and aspartate. V-ATPase-dependent acidification of the vesicles was declined by the addition of isoleucine or histidine, depending upon Avt1p. Taken together with the data of the amino acid contents of vacuolar fractions in cells, the results suggested that Avt1p is a proton/amino acid antiporter important for vacuolar compartmentalization of various amino acids.     

Characterization of CO2 flux in three Kobresia meadows differing in dominant species

Aims Kobresia meadows, the dominant species of which differ in different habitats, cover a large area of alpine grassland on the Qinghai-Tibetan Plateau and act as potential CO₂ sinks. Kobresia meadows with different dominant species may differ in carbon sink strength. We aimed to test the hypothesis and to clarify the differences in CO₂ sink strength among three major Kobresia meadows on the plateau and the mechanisms underlying these differences.Methods We measured the net ecosystem exchange flux (NEE), ecosystem respiration flux (ER), aboveground biomass (AGB) and environmental variables in three Kobresia meadows, dominated by K. pygmaea, K. humilis, or K. tibetica, respectively, in Haibei, Qinghai. NEE and ER were measured by a closed-chamber method. Environmental variables, including photosynthetic photon flux density (PPFD), air and soil temperature and air and soil moisture, were monitored during the above flux measurements.Important findings The measured peak AGB increased with soil water content and was 365, 402 and 434 g dry weight m^{-2<-sup> for K. pygmaea, K. humilis and K. tibetica meadow, respectively. From the maximum ecosystem photosynthetic rate in relation to PPFD measured during the growing season, we estimated gross ecosystem photosynthetic potential (GEP max) as 22.2, 29.9 and 37.8 μmol CO₂ m-2<-sup> s-1 for K. pygmaea, K. humilis and K. tibetica meadow, respectively. We estimated the respective gross primary production (GPP) values as 799, 1-063 and 1?158 g C m-2<-sup> year-1 and ER as 722, 914 and 1-011 g C m-2<-sup> year-1. Average net ecosystem production (NEP) was estimated to be 76.9, 149.4 and 147.6 g C m-2<-sup> year-1 in K. pygmaea, K. humilis and K. tibetica meadows, respectively. The results indicate that (i) the three meadows were CO₂ sinks during the study period and (ii) Kobresia meadows dominated by different species can differ considerably in carbon sink strength even under the same climatic conditions, which suggests the importance of characterizing spatial heterogeneity of carbon dynamics in the future.}     

Characterization of the lipids of mesosomal vesicles and plasma membranes from Staphylococcus aureus.

Mesosomal vesicles and plasma membranes were isolated from Staphylococcus aureus ATCC 6538P by protoplasting and differential centrifugation. The lipids of each of the two membrane fractions were extracted with pyridine-acetic acid-N-butanol, and the nonlipid contaminants were removed by Sephadex treatment. The lipids were then separated by passage through diethylaminoethyl-cellulose columns and characterized by thin-layer chromatographic, chemical, and spectral analyses. The lipids were separated into four discrete diethylaminoethyl fractions: (i) vitamin K2, carotenoids, C55 isoprenoid alcohol, and monoglucosyl diglyceride; (ii) cardiolipin, carotenoids, phosphatidyl glycerol, diglucosyl diglyceride, and an unidentified ninhydrin-positive component; (iii) cardiolipid and phosphatidyl glyderol; (iv) cardiolipin, phosphatidyl glycerol, and phosphatidyl glucose. Qualitatively, no difference in lipid composition between mesosomal vesicles and plasma membranes was found. However, based on equal dry weights of membrane materials, a relative quantitative difference in the amount of specific lipids in mesosomal vesicles and plasma membranes was observed. There are 4 times more monoglucosyl diglyceride, 2.6 times more diglucosyl diglyceride, 3.8 times more phosphatidyl glucose, 2 times more carotenoids, and 2 times more vitamin K2 found in mesosomal vesicles than in plasma membranes. The concentration of cardiolipin and phosphatidyl glycerol is 3.6 and 6 times greater, respectively, in mesosomal vesicles.     

Characterization of a glutamine/proton cotransporter from Ricinus comnmnis roots using isolated plasma membrane vesicles

The mechanism of glutamine transport at the plasma membrane of sink tissue cells was investigated using isolated plasma membrane vesicles from roots of Ricinus communis L. var. sanguineous . Glutamine transport was found to be driven by both the pH gradient (ΔpH) and a membrane potential (ΔΨ) (alkaline and negative internal), which were created artificially across the plasma membrane. Glutamine wus accumulated 15–20-fold in the presence of both a ΔpH and Δ Ψ . There appeared to be a direct pH effect on Δ PS -driven transport, as a higher rate of transport was observed at pH 5.5 than at pH 7.5. The ΔpH +Δ Ψ -driven transport showed saturation kinetics with a K_m of 287 μ M . Altering the membrane potential changed the V_max but had no effect on the K_m of glutamine transport. These results are consistent with the presence of a proton-coupled, carrier-mediated system for glutamine uptake in Ricinus roots. A range of protein modifiers and transport inhibitors had limited effects on glutamine transport: highest inhibition uas observed with cytochalasin D. When glutamine transport was compared in plasma membrane vesicles isolated from the root lips of Ricinus and from the remainder of the root tissue a lower level of transport was observed in the root tips. A method for the solubilization and reconstitution of glutamine transport activity using the detergent CHAPS is also described.     

Kinetics of the light-driven proton movement in model membranes containing bacteriorhodopsin.

The short-circuit photoresponse of model membranes containing bacteriorhodopsin to short (35 ms) and long (3.5 s) light pulses is described. It is shown that if the light pulse is short compared with the charging and discharging times of the model membrane, the temporal response of the light-driven proton pump can be measured. Photoactive planar model membranes were formed both from biomolecular lipid membranes and from solid 6-micrometers thick Teflon septa coated with lipid and bacteriorhodopsin. The kinetic response of the pump is independent of the planar model membrane system in which it is incorporated. Experimental evidence indicates that the shape of the leading and trailing edges of the photoresponse curve for the pump deviates from simple exponential behavior. The short-circuit photoresponse of spinach chloroplast in a planar model membrane was also studied for comparison purposes.     

Chloride flux in bilayer membranes: chloride permeability in aqueous dispersions of single-walled, bilayer vesicles.

Aqueous dispersions of phosphatidylcholine vesicles were utilized to determine bilayer permeability to 36-Cl as a function of pH and temperature. These dispersions were comprised of single-walled vesicles, homogeneous in size, prepared by sonication of purified egg phosphatidylcholine under argon followed by fractionation on a molecular sieve. Permeability constants calculated from the inward flux of 36-Cl and the geometric parameters of these vesicles proved to be dependent on both pH and temperature. Analysis of these dependences leads to the conclusion that 36-Cl permeation in the presence of KCl is due principally to a carrier mediated exchange process involving a phospholipid-HCL complex. Net permeation by H-36-Cl may make a small contribution to the 36-Cl flux, however, studies carried out at very low chloride concentrations show that this flux is much smaller than the exchange flux. Thus chloride permeability for the exchange process is 1.5 times 10- minus 11 cmsec- minus 1 while the corresponding coefficient for the net flux of H-36-Cl is 1.0 times 10- minus 12 cm sec- minus 1 at pH 7. The activation energy for the 36-Cl exchange flux was found to be 19 plus or minus 2 kcal/mol. This value is similar to that obtained for the transbilayer "flip-flop" of phosphatidylcholine molecules in a similar system (Kornberg and McConnell, 1971). This correspondence together with the fact that the experimentally determined flux of 36-Cl agrees well with that calculated from the "flip-flop" parameters, strongly suggests that the flux of 36-Cl and "flip-flop" of phosphatidylcholine may be the same process.     

Transfer of phosphatidylcholine between different membranes in Tetrahymena as studied by spin labeling.

A permeability factor was extracted in a latent form from guinea pig skin and separated by ammonium sulfate fractionation into the pseudoglobulin fraction (30--50% saturation). The activation of the latent form of the permeability factor seemed to be caused in the desalting step by gel filtration with Sephadex G-50. The factor was partially purified by streptomycin treatment and column chromatography using hydroxyapatite, diethylaminoethyl cellulose and Sephadex G-75, in this order. Gel filtration showed that its molecular weight was approx. 35000. Its permeability activity was heat stable at 61 degrees C for 60 min at neutral pH, resistant at pH 5--10 and at ionic strengths from deionized water to 1 M NaCl at 4 degrees C. Its activity was transient and suppressed by guinea pig serum, but insensitive to an anti-histamic agent (triprolidine). Furthermore, its permeability activity was inhibited by diisopropylfluorophosphate, soybean trypsin inhibitor and leupeptin, and completely adsorbed by soybean trypsin inhibitor affinity column. These findings suggested that the permeability factor was a serine-type protease.