A simple theoretical model for the effects of cholesterol and polypeptides on lipid membranes

A simple theoretical model for the effects of impurities on biomembranes is proposed. The model accounts for the cholesterol-induced decrease of membrane phase transition temperature, membrane condensation above the gel to liquid crystalline phase transition, and increase in lateral compressibility. The model also predicts that addition of molecules such as cholesterol and polypeptides to membranes results in unmasking of a continuous phase transition. This results in a second broad peak in the calorimetric curves for melting of lipid-cholesterol mixtures, and the appearance of a second melting transition in membranes modified by the incorporation of polypeptides. The theory assumes that the membrane may be adequately described by a kink model, and that impurities are randomly distributed in the membrane. The difference in size and shape of impurity molecules, compared to membrane lipids, results in a spatial disordering in the membrane which in turn causes increased chain disorder and membrane condensation, as well as a decrease in the cooperativity of melting. The second transition results from a second expansion of the condensed, partially disordered membrane, which takes place over a several degree temperature range. This transition, although unmasked by boundary effects of non-lipid molecules, does not correspond to melting of a boundary annulus or phase separation.     

Self-translocation of diphtheria toxin across model membranes.

To understand the mechanism of diphtheria toxin membrane translocation, the toxin was entrapped within lipid vesicles, and its low pH-induced translocation across the lipid bilayer was measured. Proteolysis and resistance to guanidinium chloride denaturation were used to demonstrate that the toxin molecules were entrapped. Low pH-induced movement of entrapped toxin to the outer (trans) face of the bilayer was assayed by the binding of external streptavidin to biotin-labeled entrapped toxin. Complete translocation was quantified by the amount of protein released into the external medium. Using whole toxin, it was found that the A fragment was efficiently translocated, but the B fragment was not. This was true both in the low temperature (A domain folded) and high temperature (A domain unfolded) toxin conformations previously identified [Jiang J. X., Abrams, F. S., and London, E. (1991) Biochemistry 30, 3857-3864]. Remarkably, even isolated fragment A appeared to self-translocate under some conditions. Toxin-induced translocation may partly result from formation of a nonspecific toxin-induced pore. This idea is supported by the toxin-induced release of fluorescent dextrans coentrapped within the vesicles. However, low pH-induced exposure of entrapped toxin on the outside of the membrane was conformation dependent. Exposure was greatest for the high temperature conformation. This suggests the existence of a more specific translocation process. The nature and relationship of these processes, and their relative roles in translocation in vivo are discussed.     

A kinetic analysis of the electrogenic pump ofChara corallina: III. Pump activity during action potential

Summary The current-voltage curve (I–V curve) of theChara membrane was obtained by applying a slow ramp hyper- and depolarization by use of voltage clamp. By inhibiting the electrogenic pump with 50m DCCD (dicyclohexylcarbodiimide), theI–V curve approached a steadyI–V curve within two hours, which gave thei _d -V curve of the passive diffusion channel. Thei _p -V curve of the electrogenic pump channel was obtained by subtracting the latter from the former. The sigmoidali _p -V curve could be simulated satisfactorily with a simple reaction kinetic model which assumes a stoichiometric ratio of 2. The emf of the pump (E _p ) is given as the voltage at which the pump current changes its sign. The conductance of the pump (g _p ) can be calculated as the chord conductance from thei _p -V curve, which is highly voltage dependent having a peak at a definite voltage. The changes of emf and conductance during excitation were determined by use of the current clamp (I=0). Since theE _p andg _p (V) are known, the changes, during excitation, of emf (E _d ) and conductance (g _d ) of the passive diffusion channel can be calculated. The marked increase of the membrane conductance and the large depolarization during the action potential are caused by the marked increase of the conductance of the passive diffusion channel and the large depolarization of its emf. The conductance of the electrogenic pump decreases to about half at the peak of action potential, while the pump current increases almost to a saturated level.     

A kinetic analysis of the electrogenic pump ofChara corallina: I. Inhibition of the pump by DCCD

Summary The current-voltage curve of theChara membrane was obtained by applying a slow ramp depo- and hyperpolarization by use of voltage clamp. With the progress of poisoning by DCCD (dicyclohexylcarbodiimide) theI–V curve moved by about 50 mV (depolarization) along the voltage axis, reducing its slope, and finally converged to thei _d -V curve of the passive diffusion channel. Changes ofi _p -V curve of the electrogenic pump channel could be obtained by subtracting the latter from the former.The sigmoidali _p -V curve could be simulated satisfactorily by adopting a simple reaction kinetic model. Kinetic parameters of the successive changes of state of the H⁺ ATPase could be evaluated. Changes of these kinetic parameters during inhibition gave useful information about the molecular mechanism of the electrogenic pump.Depolarization of the membrane potential, decrease of membrane conductance, and decrease of pump current during inhibition of the pump with DCCD are caused mainly by the decrease of conductance of the pump channel. The decrease of this pump conductance is caused principally by a marked decrease of the rate constant for releasing H⁺ to the outside.     

Hormone receptor mobility and catecholamine binding in membranes. A theoretical model

[³ H]-Catecholamine binding to intact cells, isolated cell membranes, and to several isolated macromolecules has been shown by several laboratories to be neither stereospecific nor inhibited by known β-antagonists. Since additional evidence indicates that this binding is not an artifact (i.e. due neither to the binding of a catecholamine oxidation product nor hormone binding to a catabolic enzyme such as COMT), the question remains as to whether this represents binding to a bona fide membrane receptor. Because all ligands which bind strongly or compete for this binding possess a catechol group, one possible explanation is that the binding affinity is primarily determined by the catechol moiety, whereas the correct stereoisomer of the side chain is necessary to activate the receptor. Thus, although binding is a necessary condition for hormone action, the necessary and sufficient condition for activation of adenyl cyclase is both the catechol group and the correct stereoisomer of the side chain. A theoretical model is developed here to provide a quantitative basis for this hypothesis. This model extends the current concept of distinct subunits in the adenyl cyclase system by separating the receptors from the catalytic sites and placing them at separate locations within the membrane. Utilizing the spare receptor model of Furchgott, and the mobility of macromolecules within a “lipid sea,” the appropriate equations to predict both hormone binding and enzyme activation are derived. Using the observed affinity constants from catecholamine binding studies, it is then shown that this model can predict the experimental observations and hence explain the apparent dichotomy arising from binding and enzyme activation studies.     

A kinetic analysis of the electrogenic pump ofChara corallina: IV. Temperature dependence of the pump activity

Summary The sigmoidal current-voltage curve (i _p -V curve) of the electrogenic H⁺-pump of theChara membrane was simulated satisfactorily with a simple reaction kinetic model which assumed consecutive changes in state of H⁺-ATPase. Four rate constants, i.e., forward and backward ones in voltage-dependent and-independent steps could be evaluated from the data. The emf of the pump (E _p ), the voltage at which the pump current changes its sign, varies only slightly with temperature. However, the pump current (i _p ) is highly temperature dependent, and there-fore the conductance (g _p ) of the pump, calculated as the chord conductance from thei _p-V curve, is also highly voltage dependent having a peak at a level somewhat less negative than the resting potential. In contrast tog _p , the conductance (i _p ) of the passive channel does not change appreciably with temperature. Arrhenius plots ofg _p and also of the rate constants showed a clear bend at about 19°C. Great temperature dependence of the kinetic parameters offers useful information on the pumping mechanism of theChara membrane.     

The Permeability of Ammonia, Methylamine and Ethylamine in the Charophyte Chara corallina (C. australis)

Ritchie, R. J. 1987. The permeability of ammonia, methylamineand ethylamine in the charophyte Chara corallina (C. australis).—J.exp. Bot. 38: 67–76 The permeabilities of the amines, ammonia (NH₃), methylamine(CH₃NH₂) and ethylamine (CH₃CH₂NH₂) in the giant-celled charophyteChara corallina (C. australis) R.Br. have been measured andcompared. The permeabilities were corrected for uptake fluxesof the amine cations. Based on net uptake rates, the permeabilityof ammonia was 6?4?0?93 µm s^–1 (n = 38). The permeabilitiesof methylamine and ethylamine were measured in net and exchangeflux experiments. The permeabilities of methylamine were notsignificantly different in net and exchange experiments, norto that of ammonia (P_methylamine = 6?0?0?49 µm s^–1(n = 44)). In net flux experiments the apparent permeabilityof ethylamine was slightly greater than that of ammonia andmethylamine (P_{ethylamine, net} = 8?4?1?2 µm s^–1 (n= 40)) but the permeability of ethylamine based on exchangeflux data was significantly higher (P_{ethylamine, exchange} =14?1?2 µm s^–1 (n = 20)). Methylamine can be validlyused as an ammonium analogue in permeability studies in Chara. The plasmalemma of Chara has acid and alkaline bands; littlediffusion of uncharged amines would occur across the acid bands.The actual permeability of amines across the alkaline bandsis probably about twice the values quoted above on a whole cellbasis i.e. the permeability of ammonia across the permeablepart of the plasmalemma is probably about 12 µm s^–1. Key words: Chara, permeability, ammonia, methylamine     

Physiological Control of Chloride Transport in Chara corallina: II. THE ROLE OF CHLORIDE AS A VACUOLAR OSMOTICUM

The extent to which Cl⁻ is replaceable as the major anionic constituent of the vacuole of Chara corallina was investigated. It was found that external Cl⁻ is not essential in order for nongrowing cells to increase internal osmotic pressure. After growth of cells in low (9 micromolar) Cl⁻, the vacuolar Cl⁻ concentration is one-half that of cells grown at normal external Cl⁻ concentration (850 micromolar). In contrast, both internal osmotic pressure and total concentration of the major cations, K⁺ and Na⁺, in the same cells were found to be only slightly sensitive to the external Cl⁻ concentration. Thus, it is proposed that, at limiting external Cl⁻ concentration, the cell is able to transport or synthesize another anion for vacuolar use rather than utilize a neutral solute.     

A kinetic analysis of the electrogenic pump ofChara corallina: II. Dependence of the pump activity on external pH

Summary The current-voltage curve of theChara membrane was obtained by applying a slow ramp de- and hyperpolarization by use of voltage clamp. By inhibiting the electrogenic pump with 50m DCCD (dicyclohexylcarbodiimide), theI–V curve approached a steady state within 100 min, which gave thei _d -V curve of the passive diffusion channel. Thei _p -V curve of the electrogenic pump channel was obtained by subtracting the latter from the former. With the increase of external pH, thei _d -V curve showed only a slight change, while thei _p -V curve of the pump channel showed almost a parallel shift, in the hyperpolarizing direction, along the voltage axis in the pH range between 6.5 and 7.5. The sigmoidali _p -V curve in this pH range could be simulated satisfactorily with the five-state model reported previously (U. Kishimoto, N. Kami-ike, Y. Takeuchi & T. Ohkawa,J. Membrane Biol. 80:175–183, 1984) as well as with a lumped two-state model presented in this report. The analysis based on these models suggests that the electrogenic pump of theChara membrane is mainly a 2H⁺/1ATP pump. The forward rate constant in the voltage-dependent step increased with the increase of external pH, while the backward one decreased. On the other hand, the forward rate constant in the voltage-independent step remained almost unchanged with the increase of external pH, while the backward one increased markedly. The pump conductance at the resting membrane potential showed either a slight increase or a decrease with the increase of external pH, depending on the sample. Nevertheless, the pump current showed generally a slight increase with the increase of external pH.     

The role of proteins in a dipole model for steady-state ionic transport through biological membranes.

The steady-state current-voltage characteristics of biological membranes are analyzed for means of an application of the electrodiffusion theory to the passage of ions through "dielectric pores", with orientable dipoles at the pore-water interfaces. A detailed evaluation of the electrostatic potential barrier shows, indeed, that the ions have practically no chance to penetrate into the phospholipid bilayer, but that they can cross the membrane through local protein inclusions, of high dielectric constant. A "gating mechanism" can be provided, moreover, by a change of the potential barrier, resulting from a dipole reorientation at the pore-water interface. Dipole-dipole interactions are opposed to the orienting effect of an applied field, but they can be neglected when the separation between the dipoles exceeds a certain critical value. The high polarizability of the pore material leads to an amplification of the effect of an applied field on the orientable dipoles. It is therefore possible to achieve a satisfactory agreement with the experimental results of Gilbert and Ehrenstein (Biophys. J., 9: 447, 1969) for the squid axon, and, in particular, to account for the width of the negative resistance regions with a relatively small value for the length of the orientable dipoles.     

Theory of excitable membranes. I. A simple model for a three-state artificial membrane.

The effect of the thermal fluctuation on the orientation distribution pattern of globular protein molecules in a two-dimensional lattice was investigated by the method of computer simulation. A set of interaction parameters was assigned to interaction sites on each molecule and the interaction energy between two molecules was given by the product of the parameters of facing sites. Orientation fluctuation was assumed to take place with the probability proportional to the Boltzmann factor. Patterns having different degrees of order appeared with the change of temperature. The entropy and other thermodynamic quantities of these patterns were calculated, and gradual and transitional changes of the pattern were discussed in comparison with the case of simple atoms or molecules.     

Spontaneous intermembrane transfer of various cholesterol-derived hydroperoxide species: kinetic studies with model membranes and cells.

Whereas spontaneous and protein-mediated transfer/exchange of cholesterol (Ch) between membranes has been widely studied, relatively little is known about the translocation of Ch oxidation products, particularly hydroperoxide species (ChOOHs), which can act as cytotoxic prooxidants. A major aim of the present study was to examine and compare the intermembrane transfer characteristics of several biologically relevant ChOOH isomers, including singlet oxygen-derived 5alpha-OOH, 6alpha-OOH, and 6beta-OOH and free radical-derived 7alpha-OOH and 7beta-OOH. These species were generated in [(14)C]Ch-labeled donor membranes [erythrocyte ghosts or unilamellar DMPC/Ch (1.0:0.8 mol/mol) liposomes] by means of dye-sensitized photoperoxidation. Spontaneous transfer to nonoxidized acceptor membranes (DMPC liposomes or ghosts, respectively) at 37 degrees C was monitored by thin-layer chromatography with phosphorimaging radiodetection (HPTLC-PI) or liquid chromatography with mercury cathode electrochemical detection [HPLC-EC(Hg)]. The former allowed measurement of total (unresolved) ChOOH along with parent Ch, whereas the latter allowed measurement of individual ChOOHs. Ghost membranes in which approximately 4% of the Ch had been peroxidized, giving mainly 5alpha-OOH, transferred total ChOOH and Ch to liposomes in apparent first-order fashion, the rate constant for ChOOH being approximately 65 times greater. Like Ch desorption, ChOOH desorption from donor membranes was found to be rate limiting, and rate varied inversely with size when liposomal donors were used. For individual ChOOHs, rate constant magnitude (7alpha/7beta-OOH > 5alpha-OOH > 6alpha-OOH > 6beta-OOH) correlated inversely with reverse-phase HPLC retention time, suggesting that faster transfer reflects greater hydrophilicity. Liposome-borne ChOOHs exhibited the same order of toxicity toward COH-BR1 cells, which are deficient in ability to detoxify these peroxides. The prospect of disseminating oxidative cell injury via translocation of ChOOHs and other lipid hydroperoxides is readily apparent from these findings.     

On the steady-state electrical potential difference across the thylakoid membranes of chloroplasts in illuminated plant cells.

The potential defference across the thyladoid membranes under steady-state saturating light conditions, measured with microcapillary glass electrodes, was found to be small as compared to the potential initially generated at the onset of illunimation. This result is discussed to be in agreement with quantitative estimates on the approximate magnitudes of the potential generating electron flux through the photo-synthetic electron transport chain and of the potential dissipating ion fluxes across the thylakoid membrane under steady-state conditions. It is concluded that a pH gradient of approx. 3-3.4 units is built up in the light across the membrane. The negative diffusion potential associated with this gradient is suggested to cause the transient negative potential observed in the dark after illumination.     

A theoretical study of the reactive dimerization of HCN.

The reaction of two molecules of HCN to produce isomeric forms of (HCN)₂ has been studied theoretically through the use of CNDO/2 calculations together with a method of energy partitioning. The present work predicts that the most stable product of such a reaction is iminoacetonitrile, and that, of the mechanisms considered in the present work, the energetically most favourable, under isolated conditions, involves the dissociation of HCN to free radicals, followed by the reaction of the CN radical with an undissociated HCN molecule and the subsequent addition of hydrogen followed by rearrangement to the imine. However, the energetics are sufficiently similar that an ionic mechanism involving CN^? might be predicted for a condensed, base-catalyzed system.     

A simple method for the simulation of steady-state diffusion through membranes: pressure-tuned,boundary-driven molecular dynamics

We present a novel molecular dynamics-based simulation technique for investigating gas transport through membranes. In our simulations, the main control parameters are the partial pressure for the components on the input side of the membrane and the total pressure on the output side. The essential point of our scheme is that this pressure control should be realised by adjusting the particle numbers in the input and output side control cells indirectly. Although this perturbation is applied sufficiently far from the membrane, the bulk-phase properties are well controlled in a simulation cell of common size. Numerical results are given for silicalite-1 membrane with permeating CH₄, CO₂, H₂ and N₂ gases as well as with binary mixtures of CO₂ with the other three components. To describe interactions between particles, we used the simple shifted and cut Lennard–Jones potential with parameters available in the literature. It is expected that the proposed technique can be applied to several other types of membranes and transported fluids in order to support the development of a deeper understanding of separation processes.     

The effect of divalent cations on Na+ tolerance in Charophytes. II: Chara corallina

Abstract The freshwater Charophyte Chora corallina dies when subjected to 70 molm^?3 NaCl if the Ca²⁺ concentration is 0.1 mol m ^?3. This stress is accompanied by a depolarization of the cell to a membrane potential more positive than E_K, a net influx of Na⁺ into the vacuole, and a net loss of K⁺ from the vacuole. Raising the Ca²⁺ concentration to 7 mol m ^?3 in the presence of elevated Na⁺ restores the Na⁺ to Ca²⁺ ratio to 10: 1 as in the control solution, and results in enhanced survival even though turgor is not regulated. Mg²⁺ is not a good substitute for Ca²⁺. It is suggested that the main reason that C. corallina fails to occupy saline habitats is its failure to regulate turgor, not sensitivity to Na ⁺, since the latter is similar to that seen in C. buckellii, which is found in saline habitats.     

The transport of chloroquine across human erythrocyte membranes is mediated by a simple symmetric carrier

The kinetic properties of the mediated transport of chloroquine in human erythrocytes are investigated. The high rates of translocation across the cell membrane and high adsorbance properties to glass surfaces have led to the development of new techniques for measuring initial rates of transport. Three different methodological procedures are used to accomplish a complete kinetic characterization of the system. All measurements were done at 25°C. Under zero-trans conditions the system displays complete symmetry, the Michaelis constants being 39.2±2.4 μM for influx and 36.6±5.6 μM for efflux. The respective maximal velocities are 206.4±36.0 μM·min^?1 and 190.0±7.8 μM·min^?1. Under equilibrium-exchange conditions the Michaelis constant is 108.6±15.6 μM and the maximal velocity is 630.3±50.4 μM·min^?1. This 3-fold increase in both K and V over the zero-trans values indicates that the rate-limiting step in the transport of chloroquine is the movement of the unloaded carrier. The kinetic data are consistent with the prediction of a simple carrier model.