A comparison of a spin-label and a fluorescent cell membrane probe using pure and mixed monomolecular films

Monocular film studies of 12-nitroxide stearic acid and 12-(9-anthroyl) stearic acid reveal that deviations from the behavior of the parent molecule (stearic acid) are as much dictated by the polar, or nonpolar, nature of the probe group as by its size. In mixed films under membrane-like conditions, the spin label probe, 12-nitroxide stearic acid, exhibits positive deviations from ideality and should read too high a fluidity. The picture is, however, complicated by a tendency of this probe molecule to adopt a bent conformation, a tendency apparently enhanced by specific interactions with the lecithin zwitterion. 12-(9-anthroyl) stearic acid, in contrast, shows only negative deviations from ideality in mixed dipalmitoyl lecithin films and should read too low a fluidity.     

Effects of temperature and cholesterol on human erythrocyte membranes

The effects of temperature and cholesterol on the membrane fluidity of human erythrocytes were studied using 5-nitroxide stearic acid (5NS), 12-nitroxide stearic acid (12NS), and 16-nitroxide stearic acid (16NS). Human erythrocytes and their lipid vesicles were treated in the range of 5--55 degrees C. In erythrocytes, ESR signals for 12NS and 16NS showed line broadening above 40 degrees C, whereas those for 5NS became sharper with increasing temperature as was the case with the signals of lipid vesicles for each label molecule. Lipid extraction from the heated sample caused no radical reduction. Only in 12NS-labeled erythrocytes did a weakly immobilized component and a strongly immobilized component appear. In the time course at 50 degrees C, the former decreased and the latter remained constant. From the ratio of both components, it was found that the interaction of the label molecules with the binding sites was determined by the physical state of the membrane. Furthermore, the dependence on temperature of the molecular motion of the labels in the cell membrane was irreversible above 40 degrees C. On addition of cholesterol to the membrane, the outer hyperfine splittings for 12NS and 16NS increased but that for 5NS decreased at C/P greater than 1, perhaps indicating a spread between the head groups of phospholipids by cholesterol.     

Effects of pH on membrane fluidity of human erythrocytes

The effects of pH on the membrane fluidity of intact human erythrocytes, ghosts, and their lipid vesicles were studied by spin label techniques in the range of pH 3.0 to 9.1. Two fatty acid spin labels, 5-nitroxide stearic acid (5NS) and 12-nitroxide stearic acid (12NS), and a maleimide spin label were used for the labeling of the membrane lipids and proteins, respectively. The outer hyperfine splitting (T parallel) was measured as a parameter of membrane fluidity. In the case of 5NS, the T parallel values for intact erythrocytes and ghosts remained almost constant over the entire pH range at 22 degrees C but those for their lipid vesicles changed slightly, indicating the vertical displacement of the labels in lipid bilayers. On the other hand, the ESR spectra of 12NS incorporated into intact erythrocytes and ghosts, as compared with their lipid vesicles, showed marked pH dependence. By means of spin labeling of membrane proteins, the conformational changes of the proteins were observed in the pH range mentioned above. These results suggest a possible association between the strong pH dependence of the T parallel values and the conformation changes of membrane proteins. The pH dependence of the membrane fluidity was also investigated in cholesterol-enriched and -depleted erythrocytes. The effects of cholesterol demonstrated that the membrane fluidity was significantly mediated by cholesterol at low pH, but not at high pH.     

Chitosan-cholesterol and chitosan-stearic acid interactions at the air-water interface

We report in this work the isotherms of cholesterol and stearic acid at the air-water interface modified by different chitosans (chitosan chloride, hydrophobic modified chitosan, and medium and high molecular weight chitosans) in the aqueous subphase. The Langmuir-Blodgett films of the complexes cholesterol-chitosan and stearic acid-chitosan are analyzed by atomic force microscopy (AFM), and a molecular simulation was performed to visualize the chitosan-lipid interactions. Strong modifications are obtained in the isotherms as a result of the chitosan interactions with cholesterol and stearic acid at the air-water interface. These modifications were dependent on the type and concentration of chitosan. Severe modifications of all phases were noticed with larger molecular areas, and the observed changes in the compressional modulus were dependent on the type of chitosan used. The complexes of chitosan-stearic acid were more flexible than the ones of chitosan-cholesterol. The AFM images demonstrated that chitosan was disaggregated by the cholesterol and stearic acid interactions producing more homogeneous surfaces in some cases. The hydrophobic chitosan showed more affinity with stearic acid, while both medium and high molecular weight chitosans produced homogeneous surfaces with cholesterol. The simulated chitosan chains interacting with cholesterol and stearic acid demonstrated the possibility of specific sites of electrostatic bonds between these molecules. Adsorption of cholesterol on the different powdered chitosans, performed by HPLC, showed that the medium and high molecular weight chitosans could retain higher proportions of cholesterol compared with the other analyzed samples.     

Influence of stearic acid monolayers upon the procaine adsorption from underlying alkaline aqueous solutions

Adsorption of procaine at the air/water interface and its penetration into stearic acid monolayers from aqueous subphase of pH 8 are studied by measuring surface tension of aqueous procaine solutions and by recording surface pressure vs. mean molecular area curves for stearic acid monolayers spread onto procaine solutions of different concentrations. The amount of procaine in the interface is derived by means of Gibbs' equation. Results are compared to those obtained earlier at pH 2 and on unbuffered subphases. With increasing pH an increasing procaine adsorption and procaine penetration is observed. This phenomenon is interpreted in terms of protolytic equilibria in which participate both surfactants procaine and stearic acid.     

Fluorescent probes in model membranes. II. monolayer studies of 2,2′-(vinylenedi-p-phenylene)bisbenzoxazole, d-3-aminodesoxyequilenin and n-octadecylnaphthyl-2-amino-6-sulfonic acid with host-lipid tetradecanoic acid

Film studies at the air-water interface have been carried out for pure films of 2,2′-(vinylenedi-p-phenylene)bisbenzoxazole (VPBO), d-3-aminodesoxy-equlenin (EQ) and N-octadecylnapthyl-2-amino-6-sulfonic acid (ONS), and for mixed films with tetradecanoic acid for the first two fluorescent probes. Pure film isotherms indicate highly rigid non-monomolecular films for both VPBO and EQ, revealing the presence of strong intermolecular forces. In mixed films with tetradecanoic acid VPBO rapidly segregates with resultant film loss over a wide concentration range. EQ, however, can be stabilized by the host-lipid at low concentrations. This, coupled with an ability to only slightly affect the host-lipid liquid-condensed/liquid-expanded phase change, suggests that EQ can be regarded as “non-perturbing” and should be retained in condensed lipid phases.ONS, because of its unusual polar headgroup, resembled hexadecanoic acid more than octadecanoic acid. While difficulties in spreading ONS precluded the study of mixed films, the indications are that it would be a satisfactory expanded lipid state probe if mixing can be brought about.     

Convenient biosynthetic preparation of isomeric spin-labelled radioactive phosphatidic acids

A convenient method for the enzymatic preparation of sn-3-[2-3H]phosphatidic acids carrying also 5-, 12-, or 16-nitroxide stearic acids, from sn-3-[2-3H]glycerophosphate and isolated guinea pig liver microsomes, is described in detail. The procedure allows a simultaneous preparation of three spin-labelled sn-3-[2-3H]phosphatidic acids of yields 3-3.5 mumol of each compound which is greater than 99% pure in respect to the radioactivity and which contains 25 mol% of spin-labelled fatty acids. These phosphatidic acids were approximately equally distributed between the primary and the secondary hydroxyl when 12- or 16-nitroxide stearic acids were used or predominantly (75%) associated with the secondary hydroxyl of sn-3-[2-3H]phosphatidic acid when 5-nitroxide stearic acid was present in the incubation mixture.     

Penetration of ascorbic acid into bilayer phospholipid membranes

Using the EPR method, the temperature dependencies of the rates of ascorbic acid-induced reduction of nitroxyl radicals carrying the nitroxyl fragment in different positions of the fatty acid chain [N(4-methylidene++-1-oxyl-2,2,5,5-tetramethyl-3-imidazolidine hydrazine)]myristic acid (I) and 1-oxyl-2,2-dimethyloxazolidine derivatives of 5-ketostearic (II) and 12-ketostearic (III) acids incorporated into egg phosphatidylcholine liposomal membranes were studied. The reduction rates, activation energy and shape of kinetic curves were found to be dependent on the mode of liposome preparation (ultrasonication or reverse phase evaporation), label type and chemical composition of the membrane (with regard to the presence or absence of stearic acid). The coefficients of partition and diffusion of ascorbic acid through the membrane lipid bilayer were calculated from the rates of transbilayer (flip-flop) diffusion of I and ascorbate penetration inside the liposomes containing Fremi salt nitroxyl radical. The experimental results formed the basis for a hypothesis on the dependence of the rate of membrane-embedded spin probe reduction on the ascorbate distribution pattern inside the lipid bilayer.     

Glutaryl phosphatidylcholine effects on pH and composition dependent behavior of liposomes studied by spin-labeling method

The influence of glutaryl phosphatidylcholine on the molecular organization of phosphatidylcholine liposomes was studied by spin-labeling technique. The ESR signals given by the 5-nitroxide stearic acid label showed that the presence of glutaryl lecithin (i) significantly increased the negative charge density of the polar liposome surface with increasing proton concentration depending on the bulk solution pH, and (ii) apparently decreased the packing (order) of the hydrophobic region close to the surface, essentially in the presence of saturated phospholipids. The spectral information--S (order parameter) and alpha N (isotropic nitrogen coupling constant)--resulted in the location of the probe near or in the polar zone of the membrane or in the hydrophobic region, depending on the protonation/deprotonation of the fatty acid carboxyl group of the probe. The microviscosity of the inner region of the membrane monitored by the 12- and 16-probes was not significantly altered by glutaryl lecithin. On the other hand, glutaryl lecithin has a lesser effect on liposomes containing anionic polar head groups, such as dipalmitoyl phosphatidylglycerol or phosphatidylinositol, the anionic charge of which already had the same effect on protonation of the polar surface. The temperature dependence of dipalmitoyl phosphatidylcholine liposome dynamic behavior indicates that the glutaryl lecithin effect is completely different above and below the gel-to-liquid crystalline phase transition point.     

Effect of high electrolyte concentration on the phase transition behaviour of DPPC vesicles: a spin label study

We have investigated by Electron Spin Resonance spectroscopy, the effects of high electrolyte concentration on the phase transitions of unilamellar vesicles of dipalmitoylphosphatidylcholine at the pH values of 5.0 and 9.0. Using the 5-nitroxide stearic acid as spin probe we have found that, at both pH values, the lipid main phase transition is not quite affected by variations of the electrolyte concentration up to the value of 3 M. Instead, the pretransition at pH 5.0 disappears in the presence of 1 M electrolyte, and at pH 9.0, the pretransition temperature shifts upward from 25.5 to 31.0 degrees C when the electrolyte concentration reaches the value of 3 M. The observed results on the pre- and main phase transition widths, transition temperatures and their cooperativity indicate that the presence of salt in the bulk solution leads to structural changes of the lipid bilayer which essentially concern either the polar zone or the hydrogen belt region of the DPPC vesicles. The extent of observed perturbation depends on salt concentration.     

Stochastic esr analysis of rat liver and hepatoma mitochondrial lipids

A commonly used model for the interaction of the motional narrowing of ESR lines is shown to be qualitatively misleading. An analysis of lipid extracts of mitochondrial preparations labeled with 12-nitroxide stearic acid produced linear plots of the logarithm of the correlation time versus the reciprocal of the absolute temperature when analyzed with stochastic computer simulations. However, when the data were analyzed with isotropic Lorentzian line shape approximations, nonlinear plots were obtained.     

Changes in lipid ordering and state of aggregation in lymphocyte plasma membranes after exposure to mitogens

Summary An electron spin probe study was made of the effect of a number of mitogenic agents on the ordering and state of aggregation of the plasma membrane lipids of lymphocytes. These agents, which included phytohemagglutinin, Concanavalin A, the calcium ionophore A23187 and periodate, caused a 20% decrease in lipid ordering in the region of the bilayer probed by 5-nitroxide stearic acid. The corresponding methyl ester probe showed marked probe-probe interaction under the same conditions indicating an aggregation of lipids in the area probed by this label. Studies with mixed lipid vesicles and gangliosidefree cells indicate that these areas are rich in glycolipids capable of hydrogen bonding to the ester probe. The decrease in ordering and the increase in aggregation of the membrane lipids were correlated with the patching and capping of the ligand-receptor complexes. Furthermore, the disappearance of fluorescent ligand from the surface of treated cells corresponded with the return of the spectral parameters of the probes to control cell values.It was concluded that glycolipids might play an important role in ligand-induced cell surface changes either as bearers of receptor groups, as in the case of some gangliosides, or in association by hydrogen-bonding with receptor proteins.     

The membrane-bound surface glycoprotein of Crithidia oncopelti

Membrane-associated surface glycoprotein from Crithidia oncopelti (MAG) has been isolated and studied. Its molecular weight constitutes 10-12 kDa. MAG contains 12% protein, 29% carbohydrates, 1.2% phosphate and a lipid fragment. Amino acid components of MAG include glutamine, asparagine, alanine, proline, valine, leucine, serine, threonine, lysine, glycine. Ethanolamine and glycerophosphate have also been found. MAG contains stearic and small amounts of palmitic, oleic and myristic fatty acids. Carbohydrate fragment of MAG consists of seven monomers.     

Effect of season on the fatty acid composition and free amino acid content of the sardine Sardinops melanostictus

The purpose of this study was to clarify the seasonal variation of fatty acid composition and free amino acid content in the Japanese sardine (Sardinops melanostictus) from the sea of Hyuga-Nada, and the relationship between the fatty acid composition of this sardine and that of plankton in the area. The lipid content of sardines at the sea of Hyuga-Nada was low in February (1.8%) and high (7.2%) from July to September. The major fatty acids in the total lipids from sardine were myristic acid (14:0), palmitic acid (16:0), stearic acid (18:0), palmitoleic acid (16:1 n-7), oleic acid (18:1 n-9), eicosapentaenoic acid (20:5 n-3), and docosahexaenoic acid (22:6 n-3). The characteristics of the fatty acids isolated from sardines in July were similar to those from plankton in the same season. This reflects the deposition of plankton fatty acids in sardine depot fat. The season of high free histidine content in the ordinary meat corresponded with that of high lipid content. These results suggested that both the fatty acid composition of sardines and the high concentrations of certain amino acids in free form are influenced by the intake and seasonal variation of composition of plankton.     

Immobilization of urease on poly(N-vinyl carbazole)/stearic acid Langmuir-Blodgett films for application to urea biosensor

Urease was immobilized in mixed monolayers of poly(N-vinyl carbazole) (PNVK) and stearic acid (SA) formed at an air-water interface. The monolayers were transferred onto indium-tin-oxide (ITO) coated glass plates using Langmuir-Blodgett (LB) film deposition technique. Urease immobilized on PNVK/SA LB films, characterized using FTIR and UV-visible spectroscopy, was found to exhibit increased stability over a wide pH (6.5-8.5) and temperature (25-50 degrees C) range. Potentiometric measurements on these urease electrodes were carried out using an ammonium ion analyzer. Two values for K(m)(app) were obtained at lower and higher concentrations of substrate urea.     

Preparation of GM1 ganglioside molecular species having homogeneous fatty acid and long chain base moieties

A new procedure is described for preparing the molecular species of GM1 ganglioside that carry a single fatty acid (myristic (C14:0), stearic (C18:0), arachidic (C20:0) or lignoceric (C24:0) acid) and a single long chain base (C18 or C20 sphingosine, C18 or C20 sphinganine, each of them in natural 3D(+)erythro or unnatural 3L(-)threo form). The procedure consisted of the following steps: a) alkaline hydrolysis of GM1 ganglioside in the presence of tetramethylammonium hydroxide, which produces de-N-acylation of the ceramide and de-N-acetylation of the sialic acid residue; b) specific re-N-acylation at the long chain base amino group with a new fatty acid (myristic, stearic, arachidic, or lignoceric) in the presence of 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride; and c) final re-N-acetylation at the level of the sialic acid residue. GM1 ganglioside molecular species, completely homogeneous in the ceramide portion, were prepared by reversed phase high performance liquid chromatography. The GM1 ganglioside molecular species were analyzed for saccharide, fatty acid, and long chain base composition by chemical and spectrometric analyses. Using a combination of the two procedures, 32 different molecular species of GM1 ganglioside, over 99% homogeneous, have been prepared.     

Quinidine and melittin both decrease the fluidity of liver plasma membranes and both inhibit hormone-stimulated adenylate cyclase activity

Increasing concentrations of either quinidine or melittin gave a dose-dependent inhibition of both the glucagon- and fluoride-stimulated activities of adenylate cyclase in the liver plasma membranes. At similar concentrations these agents increased the order of liver plasma membranes as detected by a fatty acid ESR probe, doxyl stearic acid. This increase in bilayer order (decrease in 'fluidity') is suggested to explain the inhibitory action of quinidine on adenylate cyclase activity but only in part contributes to the inhibitory action of melittin on adenylate cyclase. Arrhenius plots of fluoride-stimulated activity became non-linear in the presence of either quinidine or melittin, with a single well-defined break occurring at around 12 degrees C in each instance. Arrhenius plots of the glucagon-stimulated activity also exhibited such a novel break at around 12 degrees C when either quinidine or melittin were present as well as exhibiting a break at around 28 degrees C, as was seen in the absence of these ligands. The fatty acid spin probe inserted into liver plasma membranes detected a novel lipid phase separation occurring at around 12 degrees C when either quinidine or melittin was present and showed that the lipid phase separation occurring at around 28 degrees C in native membranes was apparently unaffected by these ligands.     

Stereospecific distribution of plamitic acid in the triacylglycerols of rat adipocytes. Effects of varying the composition of the substrate fatty acid in vitro

The effects of inclusion of different fatty acids in the medium on the rate of esterification of palmitic acid and its stereospecific distribution among the three positions of the triacyl-sn-glycerols by preparations of rat adipocytes in vitro have been determined. Myristic acid, stearic acid, oleic acid and linoleic acid were used as diluents and the concentration of the combined unesterified fatty acids in the medium was held constant; only the proportion of palmitic acid was varied. The amount of palmitic acid esterified was always linearly related to its relative concentration in the medium and was not significantly affected by the nature of the diluent fatty acid chosen. Constant relative proportions were recovered in triacylglycerols and in intermediates in each instance. The amount of palmitic acid esterified to each of the positions of the triacyl-sn-glycerols was linearly dependent on the relative proportion in the medium but the nature of the relationship was markedly influenced by which fatty acid was present. When stearic acid was present, simple relationships were found over the whole range tested. When either myristic acid, oleic acid or linoleic acid was present, abrupt changes in the manner of esterification of palmitic acid were observed in position sn-1 when the relative concentrations of palmitic acid and the diluent reached critical values, which differed with each fatty acid. In position sn-2 when oleic acid or linoleic acid was present, a similar change was observed, and in position sn-3 it was obtained with myristic acid as diluent. The results are discussed in terms of changes in the relative affinities of the acyltransferases for palmitic acid. Palmitic acid was esterified into various molecular species in proportions that indicated acylation with non-correlative specificity at higher relative concentrations but not at lower.     

Spin label studies on rat liver and heart plasma membranes: Effects of temperature,calcium, and lanthanum on membrane fluidity

The structures of rat liver and heart plasma membranes were studied with the 5-nitroxide stearic acid spin probe, I(1 2,3). The polarity-corrected order parameters (S) of liver and heart plasma membranes were independent of probe concentration only if experimentally determined low I(1 2,3)/lipid ratios were employed. At higher probe/lipid ratios, the order parameters of both membrane systems decreased with increasing probe concentration, and these effects were attributed to enhanced nitroxide radical interactions. Examination of the temperature dependence of approximate and polarity-corrected order parameters indicated that lipid phase separations occur in liver (between 19° and 28°C) and heart (between 21° and 32°C) plasma membranes. The possibility that a wide variety of membrane-associated functions may be influenced by these thermotropic phase separations is considered. Addition of 3.9 mM CaCl₂ to I(1 2,3)-labeled liver plasma membrane decreased the fluidity as indicated by a 5% increase in S at 37°C. Similarly, titrating I(1 2,3)-labeled heart plasma membranes with either CaCl₂ or LaCl₃ decreased the lipid fluidity at 37°C, although the magnitude of the La³⁺ effect was larger and occurred at lower concentrations than that induced by Ca²⁺; addition of 0.2 mM La³⁺ or 3.2 mM Ca²⁺ increased S by approximately 7% and 5%, respectively. The above cation effects reflected only alterations in the membrane fluidity and were not due to changes in probe–probe interactions. Ca²⁺ and La³⁺ at these concentrations decrease the activities of such plasma membrane enzymes as Na⁺, K⁺-ATPase and adenylyl cyclase, and it is suggested that the inhibition of these enzymes may be due in part to cation-mediated decreases in the lipid fluidity.     

A spin-label study of sciatic nerves from quaking, jimpy and trembler mice.

The spin labels, 5-nitroxide stearic acid and 16-nitroxide stearic acid were incorporated into whole sciatic nerves dissected from normal, quaking, jimpy and trembler mice. With 5-nitroxide stearic acid, we have studied the thermal variation of the maximal apparent coupling constant (T) between 0 degrees C and 50 degrees C. Within this range of temperatures, we obtained identical values of 2 T for nerves from normal and jimpy mice, whereas 2 T was smaller for nerves from quaking and trembler mice. With 16-nitroxide stearic acid, composite spectra were recorded, particularly in the high-field range. A line characteristic of myelin was clearly observed in the spectra of nerves from normal and jimpy mice; its intensity was somewhat less in nerves from quaking mice and much less in spectra from trembler mice. A shoulder in the principal highfield line of the spectrum is modified only with nerves from jimpy mice. The results agree well with those obtained by electron microscopy, which reveal normal myelination in nerves from jimpy mice, a slight modification of the myelin from those of quaking mice and a practically complete demyelination in peripheral nerves from trembler mice. However, the structure of the nerves of jimpy mice also seems to be modified at an, as yet, undetermined level.