Nuclear magnetic resonance studies of amphiphile hydration: Effects of the gel-to-liquid crystalline phase transition on the hydration of dioleoyl lecithin

The hydration of dioleoyl lecithin (DOL) and dimyristoyl lecithin (DML) has been measured as a function of temperature between ?15 and ?30 °C, using low-temperature proton magnetic resonance. The hydration of DOL is considerably higher than that of DML. We detect 9 mol of unfrozen water/mol of phospholipid at ?25 °C (our “standard” temperature) for DOL, and only 6 mol of water/mol of phospholipid for DML. The gel-to-liquid crystalline phase transition in DOL centered at ca. ?19 °C is manifested by a 70% increase in hydration for both vesicles and dispersions. Preparations of either DML vesicles or vesicles of DOL which contain 33 mol% cholesterol would not be expected to undergo this phase transition, and the hydration increase observed for these preparations in the same range of temperature is less than 20%.     

Thermotropic change in phospholipid packing in microsomal membranes sensed by phospholipase A2

A reversible, temperature-dependent change in phospholipid packing occurring between 0°C and 12°C has been identified in microsomal membranes by the use of phospholipase A₂ from Crotalus atrox. It manifests itself as a drastic increase in susceptibility to the phospholipase and depends on non-lipid (presumably protein) membrane components. It is suggested that this change could underlie the change in transmembrane mobility of phospholipids which occurs in the same temperature range.     

Prey consumption by phytoseiid spider mite predators as affected by diurnal temperature variations

The consumption rate of an ectothermic predator is highly temperature-dependent and is a key driver of pest-predator population interactions. Not only average daily temperature, but also diurnal temperature variations may affect prey consumption and life history traits of ectotherms. In the present study, we evaluated the impact of temperature alternations on body size, predation capacity and oviposition rate of the predatory mites Phytoseiulus persimilis Athias-Henriot and Neoseiulus californicus McGregor (Acari: Phytoseiidae) when presented with eggs of their natural prey, the two-spotted spider mite Tetranychus urticae Koch (Acari: Tetranychidae). For both predators, mean daily temperature as well as temperature alternation had a substantial impact on the number of prey consumed. At lower average temperatures, more eggs were killed under an alternating temperature regime (20 °C/5 °C and 25 °C/10 °C) than at the corresponding mean constant temperatures (15 and 20 °C). At higher average temperatures (>25 °C), however, the opposite was observed with higher numbers of prey killed at constant temperatures than at alternating temperatures. At 25 °C, temperature variation had no effect on the predation capacity. A similar trend as for the predation rates was observed for the oviposition rates of the phytoseiids. Body size of N. californicus was affected both by average daily temperature and temperature variation, with smaller adult females emerging at alternating temperatures than at constant temperatures, whereas for P. persimilis, temperature variation had no impact on its body size. Our results demonstrate that temperature variations are likely to affect biological control of T. urticae by the studied phytoseiid predators.     

Reversible thermal unfolding of thermostable phosphoglycerate kinase. Thermostability associated with mean zero enthalpy change.

Reversible thermal denaturation of phosphoglycerate kinases (E.C. 2.7.2.3) from an extremely thermophilic bacterium Thermus thermophilus and from yeast were studied by measuring their circular dichroism and fluorescence intensity. The thermal denaturation in the presence of guanidine hydrochloride was completely reversible. The thermodynamic parameters for the reaction were calculated based on a two-state mechanism. The free energy changes in denaturation at 25 °C in the absence of denaturant were estimated to be 11.87 ± 0.21 kcal/mol for T. thermophilus phosphoglycerate kinase and 5.33 ± 0.13 kcal/mol for that of yeast. It was found that the van't Hoff plot of the equilibrium constant for the denaturation reaction was almost independent of temperature in the temperature range 0 to 60 °C for T. thermophilus phosphoglycerate kinase, while that of yeast phosphoglycerate kinase was strongly temperature-dependent as reported for other thermolabile proteins. The enthalpy change in denaturation varies from 0.03 to 6.2 kcal/mol (0 to 60 °C) for T. thermophilus phosphoglycerate kinase and from ?27 to 31 kcal/mol (10 to 35 °C) for yeast enzyme. The entropy change in denaturation varies from ?3.9 to 21 entropy units for T. thermophilus phosphoglycerate kinase and ?96 to 104 entropys unit (10 to 35 °C) for yeast enzyme. The heat capacity change in denaturation is between 1.4 and 63 cal/deg. mol for the thermophile enzyme and between 1530 and 1750 cal/deg. mol for yeast enzyme at 20 °C. The observations that the enthalpy changes as well as the heat capacity changes in denaturation of the thermophilic enzyme were negligibly small suggest an explanation for the unusual stability to heat of T. thermophilus phosphoglycerate kinase.We also propose three possible mechanisms for the thermostability of proteins in general.     

Thermolabile Liposomes with a High Fusion Efficacy at 42°C can be Made of Dipalmitoylphosphatidylcholine/Fatty Alcohol Mixtures in the Molar Ratio of 1/2

Abstract

Liposomes made of dipalmitoylphosphatidylcholine (DPPC²), dipalmitoyl-phosphatidylglycerol (DPPG), and different long-chain fatty alcohols were investigated with respect to their colloidal stability, chain-melting phase transition temperature, and temperature dependent inter-vesicle fusion. In particular, the practical usefulness of the stoichiometric 1/2 (mol/mol) mixtures of the phospholipids and fatty alcohols, mainly elaidoyl alcohol (EL-OH) were studied. The mole fraction of DPPG in the bilayers of such vesicles affects crucially the colloidal stability of the resulting lipid suspensions; at least 15 mol-% of DPPG (relative to DPPC) must be incorporated into the bilayers in order to make the liposome suspension colloidally sufficiently stable at room temperature. The corresponding DPPC/DPPG/EL-OH (0.85/0.15/2) mixed lipid vesicles undergo a lamellar-gel to inverted hexagonal (H_IT) phase transition at 52.7°C, however, and then fuse and aggregate massively. The related phase transition temperature of the DPPC/DPPG/palmitelaidoyl alcohol (0.85/0.15/2) mixture is 48.4°C. This indicates that the chain-melting phase transition temperature of the investigated lipid mixtures is rather sensitive to the alcohol chain-length. This transition temperature is independent, however, of the bulk proton concentration in the pH region between 4.9 and 7.2. Stoichiometric 1/2 mixtures of phospholipids and EL-OH have a high propensity for the inter-vesicle fusion at 42°C and neutral pH. The reason for such fusion 10°C below the lamellar-to-nonlamellar phase transition temperature are the defects that are generated during the chain-melting of the (partly segregated) phospholipid component at 42°C; the proximity of the lamellar to non-lamellar phase transition temperature of the phospholipid/fatty alcohol (1/2) complex at 52°C also plays an important role.     

Sodium-induced aggregation of phosphatidic acid and mixed phospholipid vesicles

Sodium-induced aggregations of sonicated vesicles prepared from synthetic phosphatidic acid and from its 1 : 1 mixtures with synthetic phosphatidylethanolamine and phosphatidylcholine were studied by turbidimetric measurements. The aggregation reactions were almost completely reversible on change in the Na⁺ concentration, pH or temperature. The threshold concentrations of Na⁺ for aggregation of pure dipalmitoylphosphatidic acid vesicles and mixed dipalmitoylphosphatidylenolamine- and dimyristoylphosphatidylcholine-dipalmitoylphosphatidic acid vesicles were found to be 200, 310 and 550 mM, respectively, at 25° and pH 7.2. The hydrocarbon chain lengths of phosphatidic acid and phosphatidylethanolamine had little effect on the threshold concentrations. The threshold concentrations for phospholipid vesicles composed of phosphatidic acid alone or its 1 : 1 mixture with phosphatidylethanolamine were changed by varying either the pH or temperature, while that for phosphatidylcholine-phosphatidic acid vesicles was almost independent of the pH and temperature, implying that aggregation of the latter vesicles is induced by a somewhat different mechanism.     

Subzero temperature study of the inner mitochondrial membrane and related phospholipid membrane systems with the fluorescent probe, trans-parinaric acid

The fluorescence intensity of trans-parinaric acid as a function of the temperature indicates a phase transition in bovine heart mitochondrial inner membranes below 0°C. The comparison of the dye fluorescence intensity in intact inner mitochondrial membranes and in vesicles from extracted phospholipids of mitochondria revealed a similar intensity increase with decreasing temperature. A synthetic phospholipid system of dioleoyl phosphatidylcholine was investigated because of its low phase transition temperature and showed a very definite intensity change at ?25°C. trans-Parinaric acid in membrane systems probes an environment of intermediate polarity; this was found from the excitation and emission spectra and from fluorescence decay.     

Membranolysis by the ninth component of human complement

The lytic property of isolated C9 was shown by using heat (48°C) to induce polymerization of C9, which was then exposed to egg lecithin vesicles containing carboxyfluorescein. In this environment, C9 penetrated the vesicles and released the marker. C5b-9 and C5b-8 also released carboxyfluorescein at 48°C, as did C5b-7 to a lesser extent. However, neither isolated C5b-6, C7, C8 nor albumin caused such release. At 30°C, the C9 remained in the monomeric form and could not lyse the vesicles.C9 polymers formed at 48°C were ring-shaped with an internal diameter of approximately 100 Å and an outer diameter of approximately 180 Å. These rings of C9 polymers strikingly resembled the ultrastructures formed by the membrane attack complex of complement, viewed from the top.Our results indicate that polymeric C9 causes the membranolysis of phospholipid vesicles and, hence, that C9 alone is a cytotoxic molecule.     

Pressure-induced Shape Change of Phospholipid Vesicles: Implication of Compression and Phase Transition

A microscopic study has allowed the analysis of modifications of various shapes acquired by phospholipid vesicles during a hydrostatic pressure treatment of up to 300 MPa. Giant vesicles of dimyristoylphosphatidylcholine / phosphatidylserine (DMPC/PS) prepared at 40°C mainly presented a shape change resembling budding during pressure release. This comportment was reinforced by the incorporation of 1,2-dioleyl-sn-glycero-3-phosphatidylethanolamine (DOPE) or by higher temperature (60°C) processing. The thermotropic main phase transition (Lα to Pβ′) of the different vesicles prepared was determined under pressure through a spectrofluorimetric study of 6-dodecanoyl-2-dimethylamino-naphtalene (Laurdan) incorporated into the vesicles’ bilayer. This analysis was performed by microfluorescence observation of single vesicles. The phase transition was found to begin at about 80 MPa and 120 MPa for DMPC/PS vesicles at, respectively, 40°C and 60°C. At 60°C the liquid-to-gel transition phase was not complete within 250 MPa. Addition of DMPE at 40°C does not significantly shift the onset boundary of the phase transition but extends the transition region. At 40°C, the gel phase was obtained at, respectively, 110 MPa and 160 MPa for DMPC/PS and DMPC/PS/DOPE vesicles. In comparing volume data obtained from image analysis and Laurdan signal, we assume the shape change is a consequence of the difference between lateral compressibility of the membrane and bulk water. The phase transition contributes to the membrane compression but seems not necessary to induce shape change of vesicles. The high compressibility of the Lα phase at 60°C allows induction on DMPC/PS vesicles of a morphological transition without phase change.     

Cell surface charge and regulation of cell division of 3T3 cells and transformed derivatives.

The cell surface charge of 3T3, 3T6, SV40-3T3 cells and trypsin-, neuraminidase- and serumtreated preparations of these has been characterized by microcell electrophoresis. At 25 °C, density-inhibited 3T3 cells show a decrease in electrophoretic mobility when treated with various stimuli of cell division. This effect is not observed at 25 °C for transformed derivatives. The surface charge configuration of various cell preparations exhibits a thermal transition which is located within a temperature range characteristic of each preparation. These and other results from cell electrophoresis, taken together with those obtained in agglutination studies by other authors, are considered evidence for the occurrence in the plasma membrane of these cells of a twodimensional phase separation. The temperature range of this phase separation is shifted on treating the cells by growth stimuli. This effect might be an indication of a basic trigger mechanism in the cell cycle.     

Temperature-dependent growth and emergence of functional leaves: an adaptive mechanism in the seedlings of the western Himalayan plant Podophyllum hexandrum

As an adaptive mechanism, hypocotyl dormancy delays emergence of functional leaf until favorable season of growth in Podophyllum hexandrum, an endangered medicinal plant of the western Himalayas. However, upon exposure of the freshly germinated seedlings to favorable temperature (25°C), functional leaves emerged within 20 days. Therefore, we examined regulation mechanisms of growth and development of this alpine plant by temperature under laboratory conditions. The seedlings were exposed to (1) 25°C (temperature prevailing at the time of maximum vegetative growth), (2) 4°C (mean temperature at the onset of winter in its natural habitat), and (3) 10°C (an intermediate temperature). Slackened growth at 4°C was followed by senescence of aerial parts and quiescence of roots and predetermined leaf primordia. Rapid development of leaf primordia at 25°C was associated with increased starch hydrolysis. This was evident from higher α-amylase activity and reducing sugars. These parameters decreased on sudden exposure to 4°C. In contrast, the roots (perennating organs) showed a slight increase (1.36-fold) in α-amylase activity. Growth and development in seedlings growing at 10°C (temperature less adverse than 4°C) were comparatively faster. The content of reducing sugars and α-amylase activity were also higher in all the seedling parts at 10°C as compared to 4°C. This indicated larger requirements for sugar by the seedlings at 10°C. Irrespective of temperature, maximum changes in nitrate and nitrate reductase occurred during the initial 10 days, i.e., when the readily available form of sugars (reducing sugar) was highest. This indicated that a temperature-dependent availability of carbon, but not temperature itself, was an important regulator of uptake and reduction of nitrogen. IHBT Publication number 508a.     

Factors affecting hemagglutination by concanavalin A and soybean agglutinin

While investigating the effect of temperature on hemagglutination by concanavalin A, we noted three factors that seriously interfere with the usual microscopic agglutination assay and produce misleading or ambiguous results. (1) Adherence of concanavalin A-treated erythrocytes to surfaces of plastic Petri dishes, especially at (2) commonly used cell densities, effectively prevents determination of agglutination. (3) In addition, incubation times usually used may be insufficient to demonstrate agglutination. Failure to account for these factors may explain the previously reported temperature-sensitive, concanavalin A-mediated agglutination of trypsinized erythrocytes and transformed cells (Vlodavsky, I., Inbar, M. and Sachs, L., (1972) Biochim. Biophys. Acta 274, 364–369). By controlling these factors, we demonstrated that concanavalin A does agglutinate trypsinized, human erythrocytes equally well at 24 and 4 °C.Investigation of the kinetics of erythrocyte agglutination by lectins revealed that the rate of agglutination by concanavalin A is markedly slower at lower temperatures while soybean agglutinin-mediated agglutination is faster at lower temperatures. Ultracentrifugation data indicate that at low temperature concanavalin A exists partially as a dimer (mol. wt 50 000) and at warmer temperatures exists mainly as a tetramer (mol. wt 100 000). The correlation of the effect of temperature on molecular weight with the agglutinating activity of concanavalin A suggests that temperature-dependent forms of concanavalin A may determine the rate of cell agglutination by this lectin. No temperature-dependent change in molecular form was observed with soybean agglutinin.     

Effects of temperature and molecular interactions on the vibrational infrared spectra of phospholipid vesicles

Infrared spectra were obtained as a function of temperature for a variety of phospholipid/water bilayer assemblies (80% water by weight) in the 3000-950 cm^?1 region. Spectral band-maximum frequency parameters were defined for the 2900 cm^?1 hydrocarbon chain methylene symmetric and asymmetric stretching vibrations. Temperature shifts for these band-maximum frequencies provided convenient probes for monitoring the phase transition behavior of both multilamellar liposomes and small diameter single-shell vesiclesof dipalmitoyl phosphatidylcholine/water dispersions. As examples of the effects of bilayer lipid/cholesterol/water (3 : 1 mol ratio) and lipid/cholesterol/amphotericin B/water (3 : 1 : 0.1 mol ratios) vesicles were examined using the methylene stretching frequency indices. In comparison to the pure vesicle form, the transition width of the lipid/cholesterol system increased by nearly a factor of two (to 8°C) while the phase transition temperature remained approximately the same (41° C). For the lipid/cholesterol/amphotericin B system, the phase transition temperature increased by about 4.5° C (to 45.5°C) with the transition width increasing by nearly a factor of four ($\text{to}\text{≈ 15°}\text{C}$) above that of the pure vesicles. The lipid/cholesterol/amphotericin B data were interpreted as reflecting the formation below 38°C of a cholesterol/amphotericin B complex whose dissociation at higher temperature (38–60°C range) significantly broades the gel-liquid crystalline phase transition.     

Monoclonal antibodies to a nitroxide lipid hapten

The isolation and characterization of a hybridoma cell line producing a monoclonal IgG₁ antibody against a spin-label nitroxide group is described. The antibody recognizes a synthetic hapten containing linked dinitrophenyl and 2,2,6,6-tetramethylpiperidinyl 1-oxy groups, having an affinity of 3.6±1.0·10⁶ M^?1 for the soluble hapten at 25°C. The antibody binds to phospholipid vesicles containing 2 mol% of spin label-derivitized lipid (lipid hapten) with an affinity of 1.5±0.2·10⁸ M^?1. This monoclonal IgG₁ mediates the binding of hapten-bearing lipid vesicles to mouse macrophage RAW264 cells bearing Fc receptors. The cellular responses to this binding are similar to those observed previously using polyclonal rabbit anti-hapten IgG. As with the heterogeneous antibodies, the monoclonal IgG₁ is more efficient in mediating cellular uptake when the vesicles are in the ‘fluid’ physical state (dimyristoylphosphatidylcholine at 37°C) compared to ‘solid’ (dipalmitoylphosphatidylcholine at 37°C). Despite the enhanced binding of ‘fluid’ phospholipid vesicles to cells, only the ‘solid’ vesicles triggered a significant respiratory burst in RAW264 macrophages.     

Evidence for differently protonated forms of metarhodopsin II as intermediates in the decay of membrane-bound cattle rhodopsin.

Suspensions of vesicles from cattle rod disc membranes are flash illuminated at varying pH and temperature; the light induced proton uptake and spectral change associated with the formation of metarhodopsin II are measured. Metarhodopsin II is shown to be diprotonated at 3°C, and to exist in at least two forms at higher temperature: the diprotonated form observed at 3°C(mainly at acid pH), and an unprotonated form (mainly at alkaline pH), which is found to be in temperature dependent equilibrium with metarhodopsin I and/or protonated metarhodopsin II.     

Long N-acyl fatty acids on sphingolipids are responsible for miscibility with phospholipids to form liquid-ordered phase

The structure and thermotropic phase behaviour of aqueous dispersions of dipalmitoylphosphatidylcholine and glucosylceramide rich in C-24 fatty acyl residues was investigated by synchrotron X-ray diffraction methods. Binary mixtures comprised of molar ratios 2.5:100, 6.5:100, 12.6:100, 25:100, 40:100 and 50:100, glucolipid:phospholipid were examined in heating and cooling scans of 2°/min between 25 and 85 °C. Small-angle reflections indicated coexisting lamellar structures over the entire temperature range investigated. Reversible thermotropic changes were observed in one lamellar structure that is consistent with transitions between gel, ripple and fluid lamellar phases of pure phospholipid. The temperature of these transitions, however, were progressively shifted up by about 5 °C in the mixture containing the highest proportion of glucolipid and coincided with a published endothermic peak observed in this mixture. A higher-temperature endotherm was associated with molecular rearrangements on transition of the gel phase phospholipid to the fluid phase. This rearrangement was associated with the appearance of identifiable transient intermediate structures in the small-angle scattering region. The glucolipid formed stoichiometric mixtures with the phospholipid at all temperatures investigated and there was no evidence of phase separation of pure glucolipid. Analysis of the wide-angle scattering profiles during an initial heating scan of a binary mixture comprised of 40:60 glucolipid:phospholipid was consistent with a phase transition of pure phospholipid at about 43 °C coexisting with a liquid-ordered phase formed from the two lipids. This was confirmed by analysis of the small-angle scattering peaks of this mixture recorded at 25 and 65 °C which showed that a glucolipid-rich phase coexisted with almost pure bilayers of phospholipid at both temperatures. The glucolipid-rich phase consisted of 45:55 mole ratio glucolipid:phospholipid at 25 °C with pure phospholipid in gel phase and 42:58 mole ratio at 65 °C when the phospholipid was in the fluid phase. The results are discussed with reference to the role of the length of the N-acyl substituent of the sphingolipids in formation of complexes with phospholipids.     

Phospholipid vesicle fusion and drug loading: temperature,solute and cholesterol effects,and, a rapid preparation for solute-loaded vesicles

The fusion of sonicated dipalmitoylphosphatidylcholine (DPPC) vesicles was studied by gel-exclusion chromatography as a function of temperature, permeable and impermeable solute concentration, and cholesterol content of the bilayer membrane. Fusion is faster at lower temperatures: there is no fusion at or above 35.5°C (0.10 M DPPC/0.1 M K₂SO₄/0.01 M Hepes buffer (pH 7.4)/0.02% NaN₃). There is about 10% fusion after 1 week at 30°C, and about 60% fusion after 2 days at 13–25°C. Between 13 and 8°C, the fusion product changes from 700-Å-diameter vesicles to the 950-Å vesicles previously reported by Wong et al. (Wong, M., Anthony, F.W., Tillack, T.W. and Thompson, T.E. (1982) Biochemistry 21, 4126–4132). At 1°C, fusion is about 90% complete after 1 day. Membrane-impermeable solutes (NaCl, trehalose and glucose) inhibit fusion in a manner reflecting the total particle concentration. There is no detectable fusion after 3 days (22°C) in either 1.0 M NaCl or 2.0 M sugar, the highest concentrations studied. A suggested explanation is that impermeable solutes osmotically inhibit the influx of solution that accompanies the fusion of vesicles to form a larger vesicle, and, could conceivably thereby inhibit the fusion reaction. By contrast, membrane-permeable solutes (glycerol, ethylene glycol, propylene glycol and ethanol) dramatically increase the fusion rate. 1.0 M ethanol causes 100% fusion in 15–30 min at 22°C. The simultaneous presence of 0.15 M NaCl entirely negates the fusion-promoting effect of 1.0 M ethanol. 1 mol% cholesterol completely inhibits fusion in 0.1 M KCl (20°C), and greatly slows it down either in 1.0 M ethanol at 20°C or in 0.1 M KCl at 4°C. A suggested mechanism is that cholesterol might concentrate in and stabilize bilayer lattice defect sites that are critical for the fusion reaction. The trapping efficiency of vesicles formed by the fast ethanol fusion conditions in the presence of the water-soluble markers, chromate and arsenazo III, ranged from 9.0 to 12.7% of the marker captured in the vesicles, corresponding to trapped volumes of 1.8 to 2.5 1/mol DPPC. Bromophenol blue gave anomalously high values of 67% and 13 1/mol DPPC, which presumably reflect binding, in addition to encapsulation.     

Interaction of α-lactalbumin with dimyristoyl phosphatidylcholine vesicles. I. A microcalorimetric and fluorescence study

α-Lactalbumin and dimyristoyl phosphatidylcholine were used as a prototype to study the influence of a protein conformational change, induced by the pH, on the interaction between that protein and a phospholipid.The enthalpy changes associated with the interaction of α-lactalbumin with dimyristoyl phosphatidylcholine vesicles were measured as a function of the molar ratio of phospholipid to protein, pH and temperature. Gel-filtration, electron-microscopic and fluorescence data for the same experimental conditions were also obtained. At pH 4 and 5, the enthalphy changes (ΔH) are not only larger than at physiological pH, but also show a maximum at about 23°C in the ΔH vs. temperature graph. At pH 6 and 7, on the contrary, ΔH increases with decreasing temperature without a maximum in the curve. Gel-chromatographic and electron-microscopic data show that at pH 6 and 7, the morphological characteristics of the vesicles are unchanged upon addition of α-lactalbumin, while at pH 4 and 5 at 23°C an extra peak appears in the gel-filtration graphs between the pure vesicles and α-lactalbumin. The new fraction contains lipid-protein complexes. Electron micrographs show that bar-shaped entities are formed. A red shift at 23°C and a blue shift at 37°C, both to 336 nm, are observed for λ_max of the fluorescence emission spectra at pH 4 when α-lactalbumin is brought into contact with the phospholipid. At the same time, a strong increase in the fluorescence intensity is observed. The chromatographic and fluorescence data indicate that a lipid-protein complex with a molar ratio of approx. 80 is formed. At pH 7 and different temperatures, the emission maximum remains at the wavelength of pure α-lactalbumin, the change in the fluorescence intensity, however, indicates that interaction with the lipid occurs.The results can be explained on the basis of an electrostatic interaction at pH 6 and 7, and a hydrophobic interaction at pH 4 and 5.