Structure Of Biological Membranes Research Articles

51] Water and carbohydrate interactions with membranes: Studies with infrared spectroscopy and differential scanning calorimetry methods

Publisher Summary This chapter illustrates the ways in which two techniques, differential scanning calorimetry and infrared spectroscopy, are used to study the interactions of water with the polar head group and resulting effects of the water of hydration on the physical properties of lipids. The methods for studying the interactions of certain carbohydrates, particularly trehalose, with membrane phospholipids and proteins are discussed in the chapter. The carbohydrate, which is found at high concentrations in many organisms capable of surviving complete dehydration, is capable of stabilizing structure and function in biological membranes and preserving liposomes in the absence of water. Because of the practical implications of these properties of trehalose, the mechanism of its interaction with membrane phospholipids is emphasized in the chapter. It also describes the preparation method of omogeneous mixtures of dipalmitoylphosphatidylcholine (DPPC) and trehalose. For calorimetric measurements, Perkin–Elmer DSC 2C differential scanning calorimeter, assisted by a Perkin-Elmer 3600 data station, is used. For infrared (IR) spectroscopy, the dry preparations of lipids and membranes are either pressed into KBr disks, or observed as powder samples in thin films deposited on the windows of an aqueous IR cell with AgBr windows.

Perfluorochemicals and biological membranes: interaction and related problems

There are two probable mechanisms by which liquid perfluorochemicals (PFCs) may affect biological membrane structure and function. The first mechanism is a removal of peripheral membrane proteins and phospholipids from membrane by sorbing them on the hydrophobic perfluorinated surface. The second one is associated with PFC solubility in lipid phase of biological membranes. The solubility values for perfluorodecalin, perfluoromethylcyclohexylpiperidine, perfluorotripropylamine and perfluoro tributylamine are respectively 2.0, 0.5, 0.3 and 0.08 ml PFC per 1 kg dry erythrocyte membranes at 37°C and are in good agreement with the PFC critical solubility temperatures in hexene. Three phenomena connected with the PFC membrane solubility have been revealed: 1. Decrease of erythrocyte membrane osmotic fragility under PFC-whole blood incubation (anaesthetic-like PFCs action on membranes); 2. PFCs, soluted in the endoplasmic reticulum membranes of liver cells, form a substrate complex with cytochrome P-450 which results in the induction of drug metabolizing enzyme systems followed by activation of liver detoxic function after PFC emulsion administration to animals and human; 3. The PFC solubility in membranes controls the expiration rate constant (K ex) of PFC after injection of fluorocarbon emulsions to animals. For 7+9 PFCs formula lg K ex = a·lg(P s·C) + b has been found, where P s and C are vapour pressure and lipid solubility of PFC at 37°; a and b are constants.

Structure and dynamic properties of dehydroergosterol, 13-113-113-1

Dehydroergosterol has been widely used as a fluorescent analog of cholesterol for the investigation of lipoprotein, model membrane, and biological membrane structure. Although its synthesis was reported over fifty years ago, the complete structure and assignment of the three double bonds in the rings has not heretofore been firmly established. Therefore, dehydroergosterol was synthesized and purified by reverse phase high performance liquid chromatography. The proposed structure (Δ8, 7, 9(11), 22-ergostatetraen-3β-o1), including the location of the double bond at Δ9(11), was confirmed by mass spectroscopy,1H-NMR, and13C-NMR. In addition, a convenient assay for determination of impurities in dehydroergosterol preparations utilizing absorbance peak ratios is described. The spectroscopic properties of dehydroergosterol are highly dependent on solvent dielectric constant. Dehydroergosterol was incorporated into sonicated unilamellar vesicles composed of dimyristoylphosphatidylcholine or distearoylphosphatidylcholine. Arrhenius plots of dehydroergosterol fluorescence polarization indicated that the sterol was sensitive to the phase transitions of these phospholipids near 23° and 54°C, respectively. Differential polarized phase fluorescence and lifetime analysis were used to determine the dynamic properties of dehydroergosterol in the vesicles. At 37°C the limiting anisotropy, order parameter, and rotational rate of dehydroergosterol in dimyristoylphosphatidylcholine were 0.162, 0.65, and 0.71 nsec, respectively. The limiting anisotropy and order parameter, but not the rotational rate, of dehydroergosterol were sensitive to the temperature and/or the physical state of the phospholipid.

Biological membrane structure as "seen" by X-ray and neutron diffraction techniques.

The elastic X-ray/neutron diffraction techniques described in this review are currently capable of providing substantial information concerning the time-averaged structures and intermolecular ordering of molecular components within a dynamic membrane structure. In addition, the time resolution of the elastic X-ray diffraction technique, afforded by intense synchrotron and laser plasma X-ray sources, now permits this structural information to be obtained over a range of time scales from nanoseconds to milliseconds and upwards following an excitation of the membrane system. This time-averaged and time-resolved structural information may provide considerable insight into structure-function relationships in biological membranes and, especially when combined with structural information on the membrane proteins involved at atomic resolution, may provide this insight at the atomic level.

Dynamic structure of biological and model membranes: analysis by optical anisotropy decay measurement.

Rotational Brownian motion of molecules in membranes can be "visualized" by time-resolved measurement of the decay of anisotropy of various flash-induced optical signals, such as fluorescence, phosphorescence, delayed fluorescence, transient absorption, or fluorescence depletion. The basic principles of the various forms of anisotropy measurement are illustrated in a unified manner. In organized structures such as membranes, rotational motion is restricted in angular range. Methods of analysis of observed optical anisotropy decays for the case of restricted rotation are described; the emphasis is laid on the separate estimation of the two important parameters, the range and rate, that characterize the restricted rotation. Practical aspects of the analytical procedures are also discussed. As an example of application, recent work from the authors' laboratory is reviewed: dynamic structures of lipid hydrocarbon chain region of membranes have been revealed by time-resolved fluorescence depolarization studies. A lipophilic fluorescent probe 1,6-diphenyl-1,3,5-hexatriene was incorporated in model and biological membranes of known compositions. The decay of fluorescence anisotropy indicated that the rod-shaped probe molecules wobbled in the membranes with a wobbling diffusion constant around 0.1 rad2/nsec, presumably reflecting the dynamics of surrounding lipid chains. The effects of temperature, ions, lipid chain unsaturation, cholesterol, and protein on the range and rate of wobbling were examined with model membranes. The dynamic structure of biological membranes was found to be basically similar to that of the bilayer of unsaturated phospholipid; proteins and cholesterol act mainly as barriers that reduce the angular range of wobbling motion.

Preparation of frozen-hydrated specimens for high resolution electron microscopy

A method is presented for preserving the high resolution structure of biological membranes in a frozen-hydrated environment for electron microscopy. The technique consists of sandwiching a specimen between two carbon films and then waiting while some of the solvent evaporates. When the solvent layer is judged to be of an appropriate thickness, the specimen is then frozen in liquid nitrogen. The specimen can then be inserted into the precooled stage of an electron microscope. Electron diffraction studies of the purple membrane of Halobacterium halobium recorded at — 120°C have shown that the structure can be preserved to a resolution of 3.5 Å. The main advantage of this method over previous techniques is that the hydrating conditions can be accurately controlled.

Observation of Concanavalin-A receptors on hydrated planar erythrocyte membrane film by in situ electron microscopy

Monolayers and planar "black" lipid membranes have been widely used as models for studying the structure and properties of biological membranes. Because of the lack of a suitable method to prepare these membranes for electron microscopic observation, their ultrastructure is so far not well understood. A method of forming molecular bilayers over the holes of fine mesh grids was developed by Hui et al. to study hydrated and unsupported lipid bilayers by electron diffraction, and to image phase separated domains by diffraction contrast. We now adapted the method of Pattus et al. of spreading biological membranes vesicles on the air-water interfaces to reconstitute biological membranes into unsupported planar films for electron microscopic study. hemoglobin-free human erythrocyte membrane stroma was prepared by hemolysis. The membranes were spreaded at 20°C on balanced salt solution in a Langmuir trough until a surface pressure of 20 dyne/cm was reached. The surface film was repeatedly washed by passing to adjacent troughs over shallow partitions (fig. 1).

Book Reviews

Book Review in this Article:Nerve Membrane, Biochemistry and Function of Channel Proteins: Proceedings of the 5th Taniguchi International Symposium in Biophysics, Structure, and Function of Biological Membranes edited by Gen Matsumoto and Masao Kotoni.Biological Aspects of Schizophrenia and Addiction edited by G. Hemmings.Thiamin: Twenty Years of Progress. Annals of the New York Academy of Sciences, Vol. 378 edited by Henry Z. Sable and Clark J. Gubler.

Nuclear magnetic resonance line-shape analysis of fluorine-19-labeled phospholipids.

The application of a fluorine-19 probe to the problem of motions present in the hydrophobic region of phospholipid dispersions and biological membranes has been extended to the study of phospholipids labeled with fluorine-19 in the 8 position and with deuterium in the 2, 7, and 9 positions of the 2-acyl chain. 1-Myristoyl-2-(8,8-difluoro[2,2,7,7,9,9-2H6]myristoyl)-sn-glycero-3- phosphocholine and its nondeuterated analogue have been investigated by 19F nuclear magnetic resonance at 282.4 MHz. Spectra obtained from macroscopically oriented bilayers exhibit Pake doublets from which order parameters can be obtained. The spectra obtained from nonoriented liposomes of the phospholipids can be explained in a satisfactory manner as a random superposition of doublets broadened by heteronuclear magnetic dipole-dipole interactions. From an analysis of the data, several conclusions about the motional state of the hydrocarbon chains in the liquid-crystalline phase can be drawn. The present results show that appropriate fluorine-19 probes in the acyl chains of phospholipids can be used to investigate the structure and dynamics of model and biological membranes.

The hydrophobic interaction is long range, decaying exponentially with distance.

The attractive interaction between organic nonpolar molecules, such as hydrocarbons, in water is unusually strong. This ‘hydrophobic interaction’1 is responsible for the very low solubility of hydrophobic molecules in water, and has a central role in micelle formation, biological membrane structure, and in determining the conformations of proteins2,3. It was once believed that because the interaction is so strong there is a ‘hydrophobic bond’ associated with it2,4; but it is now recognized that the interaction involves the configurational rearrangement of water molecules as two hydrophobic species come together5–9 and is therefore of longer range than a typical covalent bond. However, there has been no experimental information available concerning the distance dependence and effective range of this interaction. From measurements of the total force as a function of distance between two hydrophobic surfaces immersed in aqueous electrolyte solutions we have determined accurately the attractive component due to the hydrophobic interaction and found that the hydrophobic interaction has the same range as, but is about an order of magnitude stronger than, the van der Waals-dispersion force; and that in the range 0–10 nm it decays exponentially with distance with a decay length of ∼1 nm. The results can be roughly extrapolated to molecular interactions and show that the interaction free energy of two hydrophobic solute molecules of radius R (nm) in water at 21 °C is approximately given by ΔGH = −40R kJ mol−1, which is in agreement with previous estimates. However, the hydrophobic interaction is not due to a ‘hydrophobic bond’, and its long-range nature has obvious implications for the mechanism and rates of folding as well as the equilibrium conformations of proteins and other macromolecules.

Organization of the glycosphingolipid asialo-G M1 in phosphatidylcholine bilayers

An affinity purified monovalent ferritin conjugate of Ricinus communis agglutinin (RCA 60) is used with freeze-etch electron microscopy to study the ultrastructural localization of the glycosphingolipid asialo-G M1 in multilamellar phosphatidylcholine liposomes. Dimyristoylphosphatidylcholine (DMPC) liposomes containing up to 20 mol% asialo-G M1 and quenched below the main transition temperature show a striking linear localization of ferritin-RCA 60 between phospholipid ridges. The glycosphingolipid localization is similar to that postulated for up to 20 mol% cholesterol in pure phosphatidylcholine bilayers by Copeland, B.R. and McConnell, H.M. (Biochim. Biophys. Acta, 599, 95–109 (1980)). Above the main phase transition temperature, asialo-G M1 appears to be organized into clusters, especially in palmitoyloleoylphosphatidylcholine (POPC) liposomes. This clustered distribution of glycosphingolipids seen above the phase transition temperature suggests that this type of lipid may exhibit compositional domain structure in biological membranes.

Investigation of membrane structure using fluorescence quenching by spin-labels. A review of recent studies.

Fluorescence quenching is the loss of fluorescence intensity which is observed when a fluorescent molecule or group interacts with another molecule or group, called the quencher. By use of tryptophan residues of proteins, together with specific probe molecules, quenching can be applied to problems of biological and model membrane structure. Quenching interactions are short range (less than 50 A) so that structure on the scale of molecular dimensions can be examined. This review summarizes the recent applications of fluorescence quenching by spin (nitroxide)-labeled molecules to problems of membrane structure, including determination of the distance of membrane-bound molecules from the membrane surface, the strength of lipid-protein interactions and the strength of protein-protein interactions within membranes. The unique advantages and the limitations of this powerful method are examined.

Structural analysis of membranes by means of a nuclear reaction

A narrow nuclear resonance in low-energy proton-induced nuclear reactions in the stable isotope 18O has been utilized in a new technique for investigating the structure of biological membranes. This technique leads to a direct determination of the density profile of 18O-labelled molecules. Results with a multilayer of egg lecithin and with erythrocyte ghosts demonstrate the applicability of the method to structural analysis.

A review of lecithin chemistry and glandless cottonseed as a potential commercial source

AbstractIndustrial lecithin can be fractionated as phospholipids and glycolipids after neutral lipids and protein‐containing contaminants are removed. The polar lipids are very reactive and are difficult to extract and purify from oilseeds. Their purity and special properties can be improved by a number of methods including solvent fractionation, hydrogenation, sulfonation, and ethoxylation. Studies are determining the role of the polar lipids of lecithin in (a) the synthesis of triglycerides in maturing seeds, (b) the structure of biological membranes, and (c) the molecular basis of the functionality of food ingredients. Lecithin, having both polar and nonpolar groups, has high surface activity and is reactive with both oil and protein, making it an excellent emulsifying agent in food systems; lecithin also slows autoxidation and enzyme hydrolysis of fats. Cottonseed lecithin is low in linolenic acid, prevents flavor deterioration of soybean oil and can be used to stabilize sunflower oil against color change during high temperature use. Gossypol binds to lecithin in oil from glanded cottonseed economically negating it as a commercial source of this product. New cultivars producing glandless, or gossypol‐free cottonseed, may have potential as commercial sources of edible lecithin.

Dynamic structure of biological membranes as probed by 1,6-diphenyl-1,3,5-hexatriene: a nanosecond fluorescence depolarization study.

A fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene, was incorporated in four different biological membranes, the purple membrane of Halobacterium halobium, human erythrocyte membrane, rabbit sarcoplasmic reticulum membrane, and rat liver mitochondrial membrane. Time-resolved fluorescence depolarization of the probe suggested that the rotational Brownian motion of the probe in the membranes was restricted in the angular range. The motion of the rod-shaped, lipophilic probe molecule, expected to reflect closely the motion of neighboring lipid hydrocarbon chains, was analyzed in terms of the wobbling-in-cone model in which the major axis of the probe was assumed to wobble freely in a cone of semiangle theta c with a wobbling diffusion constant Dw. At 35 degrees C, Dw in the four membranes, in the above order, ranged between 0.048 and 0.15 ns-1 and theta c between 31 and 53 degrees. From the rotational rate Dw, the viscosity against the wobbling motion was calculated to be 0.9-0.3 P. When the temperature was raised from 10 to 35 degrees C, Dw in all membranes increased approximately 3-fold, corresponding to activation energies of 7-8 kcal/mol, and theta c increased by about 10 degrees, except for the purple membrane in which the angular range remained narrow. The same characteristic temperature dependence has been found in many model membrane systems that contain unsaturated lecithins, suggesting an important role of unsaturated phospholipids in the dynamic structure of the lipid hydrocarbon chain region of biological membranes at physiological temperatures. Comparison with model systems suggests that proteins and cholesterol act mainly as barriers that narrow the angular range.

Structure of Biological Membrane and Its Model

Structure of Biological Membrane and Its Model

膜トピックス

The recent situation of research and development on the following items is surveyed:(1) The characteristics (structure and functions) of biological membranes and synthetic membranes.(2) Materials, structures and shapes of the various kinds of the membrane separator.(3) Artificial organs-Partial substitution of some biological functions by synthetic polymers, for example, skin, eye, lung and kidney.(4) A role of synthetic membranes for use in the systems of blood component separation, blood purification and plasma exchange.(5) A co-operation of the artificial membrane with the biological substances - A cell culture on the surface of the synthetic membranes.

Electron spin resonance: spin labels.

In general biological membranes possess no intrinsic paramagnetism and hence in the unlabeled state do not give rise to an electron spin resonance (ESR) spectrum. The introduction of a stable free radical (“spin label”) thus enables one to use ESR spectroscopy to study specific environments within the membrane. The spin label which is invariably used is the nitroxide radical, which has a three-line nitrogen hyperfine structure whose splitting varies with the orientation of the magnetic field relative to the nitroxide axes. It is this spectral anisotropy which has made spin label ESR such a powerful tool in the study of the molecular motions which are the characteristic feature of the highly dynamic structure of biological membranes. The nitroxide hyperfine splittings are partially averaged by the anisotropic motion, which gives a measure of the motional amplitude, and the linewidths are differentially broadened by an extent which depends on the rate of molecular motion. Other important features of the spin label spectra are the broadening by intermolecular label-label interactions and the ability to detect compartmentation of the label by quantitating spectral lineheights after selectively removing accessible spin-label signals by chemical reducing agents. Label-label interactions arise from two sources: the Heisenberg exchange interaction which is essentially a contact interaction and is therefore capable of measuring translational diffusion; and the dipole-dipole interaction which depends on the distance apart of the labels and is therefore capable of measuring intermolecular separations. Quantitation after treatment with reducing agents is chiefly concerned with the measurement of transport properties: of spin-label substrates or reducing agents, or the translocation of labelled lipid molecules.

Hepatic plasma-membrane modifications in disease.

The idea that aberrant cell behaviour can be pinpointed to critical molecular modifications has gained elegant experimental support in studies showing specific nucleic and corresponding amino acid changes in inherited disorders in haemoglobin synthesis and structure. The realization that many physiological processes are membrane mediated, coupled with progress in knowledge of the structure and function of biological membranes, has encouraged many investigators to study membrane pathology. The plasma membrane is positioned strategically for modification by intraand extracellular factors. The easy availability and relative simplicity of erythrocyte membranes has already led to the demonstration of membrane changes in genetic and infectious diseases, and two recent examples include those detected in haemoglobinuria (Banga, Pinder, Gratzer, Linch & Huehns, 1979) and aRcr infection by malaria sporozoites (Wallach, 1979). The plasma membrane of nucleated cells, and especially those constituting tissues and organs, is a far more complex organelle. In addition to its various transport functions, the plasma membrane has been shown to be a primary site of action of polypeptide hormones, viruses, drugs and toxins. The plasma membrane’s outer surface is also a key component in controlling cell-cell interactions, as well as cell, organ and tissue specificity. The metabolic versatility of the hepatocyte is reflected at its cell surface, where the plasma membrane is differentiated into at least three major anatomical and functional regions. The plasma membrane at the blood-interfacing sinusoidal and bile-interfacing canalicular regions forms microvilli and these two regions are separated by smooth

A new approach to the study of lipid-protein interactions in the structure of biological membranes

The lytic action of a number of N-acyl amino acids on lecithin liposomes was examined. The agents' affinity for the lecithin liposome membrane was measured and the results obtained were treated to estimate the interactions of the amino acid residues with the lecithin polar head group at the surface of the liposome membrane. The data were considered in relation to the study of the surfactant effects on the erythrocyte volume. The ability of the suggested approach to obtain information on protein-lipid interactions inaccessible by other techniques is briefly commented on.