I.R. Miller

Perturbations of membrane structure by optical probes: I. Location and structural sensitivity of merocyanine 540 bound to phospholipid membranes

SummaryThe maximum monomer absorption wavelength of a frequently used external membrane probe, Merocyanine 540, can be related to the location of the binding site for the dye within lipid membranes. Solvent studies indicate the occurrence of very specific and mutual perturbances between the probe and its microenvironment, that are of relevance, when investigating structural and functional events in biomembranes with the aid of this dye. Merocyanine 540 (MC 540) is an excellent probe for structural altions in the lipids including phase transitions. The extinction coefficient and λmax place the location of the dye-chromophore slightly above the domain of the glycerol of backbone of neutral and charged phospholipids. This explains the sensitivity of MC 540 to structural variations in the head-group region of several synthetic dipalmitoyl-lecithin analogues. The major physical parameters involved in variations of the optical signals associated with changes in the membrane structure are the dye/lipid partition coefficient and the monomer-dimer dissociation constant of the dye bound to the lipids. A temperature dependent transition from the liquid-crystalline to the crystalline state leads mainly to an exclusion of the dye from the lipid phase with a concomitant dimerization of the dye molecules still in contact with the polarhead group region of the lipid. The relevance of this finding for the mechanism of transient optical signals in connection with the occurrence of action potentials in excitable membranes is discussed. Our findings underline the necessary caution when applying external optical probes and analyzing membrane features from the spectral data, because of inevitable perturbances in the microenvironment of every probe molecule.

Compositional aspects of lipid hydration.

The effect of various lipids such as cerebrosides, gangliosides and dipalmitoyl lecithin (DPPC) on the lowering of the melting temperature of water, was determined by differential scanning calorimetry (DSC). The lowering of the melting temperature, and the number of water molecules per lipid molecule which apparently do not undergo melting, increase with the increase of the size and charge of the polar group and with the unsaturation of the hydrocarbon chains. Freezing curves show supercooling to about -20 degrees C. It was found that the number of apparently unfreezable water molecules is about four for glucocerebroside from Gauchers spleen and about eight or nine for galactocerebroside from bovine brain. In gangliosides from bovine brain the following number of water molecules/lipid molecule are apparently unfreezable: 22-30 in GM1, 33-40 in GD1a + GD1b while a fraction of gangliosides containing 75% GQ1b and 25% GT1b affects up to 60 molecules of water/molecule of lipid. A zwitterionic DPPC molecule removes apparently six to seven water molecules from freezing. There is no indication that the apparently unfreezable water molecules are in a distinct state. It is suggested that they freeze at very low temperatures producing a flat tail preceding the transition peak which cannot be discerned from the base line.

Calorimetric studies on various gangliosides and ganglioside-lipid interactions

Differential scanning calorimetry was used to investigate the thermotropic behaviour of various gangliosides differing in size and in the net negative charge. It was found that the number and the position of the negative charges in the headgroup region influence strongly the phase transition profiles. Interaction of GM1 ganglioside with egg phosphatidylcholine or cholesterol was also investigated. GM1 is completely miscible with egg phosphatidylcholine, giving only one transition peak at all ratios of the two components, implying that when gangliosides are in a more fluid lipid environment in biological membranes they will be randomly distributed. Interaction with cholesterol decreases the enthalpy of melting of the ganglioside. The decrease in enthalpy reaches a plateau at about 30 mol% cholesterol, suggesting a lower affinity of cholesterol for gangliosides than for sphingomyelin.

Selective binding of polymyxin B to negatively charged lipid monolayers

Abstract It is demonstrated by direct measurement of surface radioactivity that the cationic polypeptide antibiotic polymyxin B is specifically adsorbed to negatively charged lipid monolayers. The latter attracted the following amounts of the biologically active mono -N[ 14 C] acetylpolymyxin B derivative (PX): lipid A from Proteus mirabilis, 0.17; phosphatidic acid, 0.12; phosphatidylglycerol and phosphatidylserine, 0.11; dicetylphosphate, 0.107; sulfoquinovosyldiglyceride, 0.104; phosphatidylinositol and cardiolipin, 0.095; and phosphatidylethanolamine, 0.017 μg/cm2. Adsorption of PX to phosphatidylcholine, monogalactosyldiglyceride and stearylamine was almost or completely zero. Total lipids from Escherichia coli adsorbed 0.057 in comparison to 0.051 μg PX/cm2 of an artificial mixture of phosphatidylethanolamine/phosphatidylglycerol/cardiolipin in the proportions 75 : 25 : 5. The concentration of the surface active PX at the air/water interphase was 0.091 μg/cm2. These saturation surface concentrations of PXat lipid monolayers were reached at 1 μg/ml bulk concentrations in 2 mM NaCl/1 mM Tris · HCl, pH 7.2. They decreased with decreasing surface charge density of the adsorbing monolayer. In an experiment with cardiolipin/phosphatidylethanolamine mixtures it was shown that two molecules of cardiolipin induced adsorption of one molecule PX giving a 1 : 1 ratio with regard to positive and negative charges. This could be due to a similar charge density of about one charge per 40–50 A2 in PX and lipid bilayers composed of phospholipids. The electrostatic PX-lipid interaction was severely inhibited by 10−2 and 10−1 M Ca2+ and Na+, respectively. It is discussed that the specificity of PX against Gram-negative bacteria is caused by the occurrence of lipid A, phosphatidylglycerol and cardiolipin at the cell surface of these microorganisms.

The mechanism of electroreduction of cystine at the mercury electrode

Summary Potential sweep oscillopolarography of solutions containing cystine and cysteine has been carried out. Mercury cysteinate is formed on the mercury electrode surface in contact with either cystine or cysteine solution at potentials more positive than the reduction potential of cystine. When a cathodic sweep is then applied, the adsorbed mercury cysteinate is reduced, producing a reversible reduction peak. The formation of mercury cysteinate from cysteine is always fast, but its formation from cystine is very slow at low pH values and its rate increases with increasing pH. The mercury in the mercury cysteinate may be in the monovalent or bivalent form but higher surface concentrations of cysteine favor the bivalent form. Heavy metal ions tend to displace mercury from mercury cysteinate shifting the reduction peaks to potentials more negative than that of mercury cysteinate or of the pure heavy metal ion.

The molecular composition of some lipid bilayer membranes in aqueous solution

SummaryAn analytical technique is described for direct determination of the molecular composition of lipid bilayer membranes in aqueous solution. Membranes formed from chemically pure, radioactively labeled components, were sampled by pipetting a mercury droplet through the bilayer-water interface. During this procedure, the membrane remains intact but decreases in area with a concomitant increase in the area of the surrounding bulk phase. It is shown that each mercury droplet is covered with a fragment of the bilayer membrane in the form of a closed vesicle. The chemical composition of the bilayer is determined from an analysis of the readioactivity on the mercury droplet.Bilayers generated from glyceryl monooleate inn-decane orn-hexadecane contain (4.7±0.4)×1014 molecules of monoglyceride per cm2 and a minimum of (2.8±0.7)×1014 molecules of solvent (n-hexadecane) per cm2. It is estimated from these numbers that 37 vol % of the hydrocarbon core of the bilayer is occupied by solvent.The composition relationships between the bilayer and bulk membrane-forming solution were determined for mixtures of glyceryl monooleate (GMO) with cholesterol (Chol) or glyceryl monostearate (GMS). It was found that [GMS/GMO]bilayer≃[GMS/GMO]bulk, and [Chol/GMO]bilayer=0.5[Chol/GMO]bulk. While the molecular areas of glyceryl monooleate and glyceryl monostearate are unchanged in the mixed system, the average area for mixtures of cholesterol and glyceryl monooleate is decreased, suggesting a condensing effect of the sterol in the bilayer analogous to that observed in lipid monolayers.

Transport of ions across lipid monolayers: II. Reduction of polarographic currents of Cu(II) by decylammonium monolayers

Abstract The rate of transport of Cu (II) ions across a decylammonium monolayer was measured as a function of decylamine and salt concentration and also of potential using the polarographic technique. Utilizing information about the structure of the monolayer, it is possible to give the transport results in terms of molecular parameters. For constant salt concentration, the monolayer permeability to ions decreases with an increase in monolayer concentration and surface pressure. The effects of varying salt concentration on the permeability are somewhat complicated because of simultaneous changes in surface concentration and pressure. Some unusual humps in the polarogram appear to be due to the adsorption of micelles above the critical micelle concentration. The data were analyzed in terms of transport rate constants for crossing the monolayer on the basis of a model due to Koutecky. An expression for the rate constant was derived in terms of absolute rate theory and an activation area was estimated on this basis. The derived rate constant helps to explain a number of aspects of the transport process including the conclusion that the transported ionic species is actually monovalent.

Adsorption of ribonuclease at the air-water interface and on phospholipid monolayers

Abstract Adsorption of ribonuclease at the air-water interface and on lecithin and cephalin monolayers was followed using a tritiated acetic anhydride-labeled protein. At the air-water interface, the ribonuclease was adsorbed in two distinctly different and not necessarily monomolecular layers. The first layer several molecules thick seemed to be completely or partly composed of unfolded protein molecules. The second layer seemed to be composed of native ribonuclease molecules in a bidimensional crystalline arrangement. On a condensed phospholipid monolayer, only a one-layer adsorption of native ribonuclease molecules was obtained. When the phospholipid monolayer was expanded, the adsorption characteristics of the ribonuclease on it became similar to those at the air-water interface.

Vesicles from mixtures of bipolar archaebacterial lipids with egg phosphatidylcholine

Bipolar lipids from the membranes of archaebacterium Caldariella acidophila can form small unilamellar liposomes, when sonicated from lipid mixtures containing at least 25 mol% egg phosphatidylcholine. With increasing contents of archaebacterial lipid the inner radius of highly sonicated vesicles increases (from approx. 90 A to approx. 160 A) concomitant with an enhanced asymmetric distribution of the phosphatidylcholine molecules towards the outer face of the lipid bilayer membranes.

Transport of ions across lipid monolayers. IV. Reduction of polarographic currents by spread monolayers

Abstract The equilibrium distribution of spread lipid monolayers between the air/water and the polarized mercury/water interface has been determined. The technique involved measuring the concentrations of 3 H-labeled monolayer constituents on the surface of mercury drops which passed through the interface from a dropping mercury electrode located above the air/water interface. The same dropping mercury electrodes have been used to determine the differential capacitance in the presence of these monolayers and the permeability of the monolayers to different ions [Cu 2+ , Ag + , Tl + ). The permeability to the different ions, as measured by polarographic currents, decreases with the compression of the liquid (e.g., oleyl alcohol and oleic acid) monolayers, and is lowest around the zero charge potential at the mercury surface. High positive or negative polarizations of the mercury electrode cause a reorientation and an expansion of the monolayer at the mercury/water interface with a consequent increase in permeability and differential capacitance. Addition of low mole fractions of negatively charged molecules or crown ethers to the monolayer enhances ion permeability, while addition of cholesterol, which has a condensing effect on the monolayer, reduces ion transport. If the mole fraction of the cholesterol added to the monolayer exceeds 0.5 the homogeneity of the monolayer breaks down and its permeability increases sharply.