Showing papers on "Membrane published in 1981"

Glycosphingolipids in Cellular Interaction, Differentiation, and Oncogenesis

Abstract: The idea that glycosphingolipids (or, briefly, glycolipids) are ubiquitous components of plasma membrane and display cell type-specific patterns perhaps stemmed from the classical studies on glycolipids of erythrocyte membranes.(1,2) Subsequently, plasma membranes of various animal cells were successfully isolated and analyzed; all were characterized by their much higher content of glycolipid than was found in intracellular membranes.(3–8) It is generally assumed that glycolipids are present at the outer leaflet of the plasma membrane bilayer, although this assumption is based only on experiments with surface-labeling by galactose oxidase-NaB[3H]4 of intact and lysed erythrocyte membranes and inside-out vesicles.(9,10) Obviously, further extensive studies of the organization of glycolipid in other eukaryotic cell membranes are necessary. Interestingly, the majority of neutral glycolipids present in plasma membranes are cryptic (see Section 4.2.1).

Ca-dependent K channels with large unitary conductance in chromaffin cell membranes

Abstract: Injection of Ca ions increases the membrane permeability of many types of cells (reviewed by Meech1), and in molluscan neurones, removal of Ca ions from the external solution suppresses a voltage-dependent K conductance2,3. These two phenomena may be related through a potential–dependent Ca influx2. The question then arises whether the voltage dependence of the corresponding K permeability system is an intrinsic property of this system or whether it is merely a consequence of the increase of Ca influx with depolarization. The resolution of this and similar problems has been hampered by technical difficulties, due to the presence of voltage–dependent Ca channels, of Ca–independent K conductances and of strong intra-cellular buffering for Ca ions. In the present study patches of membranes containing functional, Ca-dependent K channels have been isolated from chromaffin cells. It is shown that application of low Ca concentrations to the inner side of the membrane affects the properties of the channels, while Ca ions are ineffective on the outer side of the membrane. The method used here overcomes several of the limitations of the previous studies using cellular recording.

Cation and Water Diffusion in Nafion Ion Exchange Membranes: Influence of Polymer Structure

Abstract: Membrane self‐diffusion coefficients for sodium ion, cesium ion, and water have been measured for Nafion® 120 perfluorosulfonate ion exchange membranes. Values have been determined as a function of temperature, and as a function of membrane water content by studying samples in heteroionic forms. The diffusional properties of this polymer are found to differ from those of conventional polystyrenesulfonates in several respects, and the free volume theory which describes ionic diffusion in the latter is inappropriate to treat Nafion. Results indicate that cations may exist in two distinct regions in the polymer; the proportion of total cations in each region may depend on the ions size and charge density. A structural model of Nafion, which correlates the membrane's spectroscopic and diffusional properties, is proposed to explain the results.

Adsorption of divalent cations to bilayer membranes containing phosphatidylserine

Abstract: The Stern equation, a combination of the Langmuir adsorption isotherm, the Boltzmann relation, and the Grahame equation from the theory of the diffuse double layer, provides a simple theoretical framework for describing the adsorption of charged molecules to surfaces. The ability of this equation to describe the adsorption of divalent cations to membranes containing brain phosphatidylserine (PS) was tested in the following manner. Charge reversal measurements were first made to determine the intrinsic 1:1 association constants of the divalent cations with the anionic PS molecules: when the net charge of a PS vesicle is zero one-half of the available sites are occupied by divalent cations. The intrinsic association constant, therefore, is equal to the reciprocal of the divalent cation concentration at which the mobility of a PS vesicle reverses sign. The Stern equation with this association constant is capable of accurately describing both the zeta potential data obtained with PS vesicles at other concentrations of the divalent cations and the data obtained with with vesicles formed from mixtures of PS and zwitterionic phospholipids. Independent measurements of the number of ions adsorbed to sonicated PS vesicles were made with a calcium-sensitive electrode. The results agreed with the zeta potential results obtained with multilamellar vesicles. When membranes are formed at 20 degrees C in 0.1 M NaCl, the intrinsic 1:1 association constants of Ni, Co, Mn, Ba, Sr, Ca, and Mg with PS are 40, 28, 25, 20, 14, 12, and 8 M-1, respectively.

Inorganic mercury (Hg2+) transport through lipid bilayer membranes

Abstract: Diffusion of inorganic mercury (Hg2+) through planar lipid bilayer membranes was studied as a function of chloride concentration and pH. Membranes were made from egg lecithin plus cholesterol in tetradecane. Tracer (203Hg) flux and conductance measurements were used to estimate the permeabilities to ionic and nonionic forms of Hg. At pH 7.0 and [Cl−] ranging from 10–1000mm, only the dichloride complex of mercury (HgCl2) crosses the membrane at a significant rate. However, several other Hg complexes (HgOHCl, HgCl 3 − and HgCl 4 2− ) contribute to diffusion through the aqueous unstirred layer adjacent to the membrane. The relation between the total mercury flux (J Hg), Hg concentrations, and permeabilities is: 1/J Hg=1/P ul[Hg t ]+1/P m [HgCl2], where [Hg t ] is the total concentration of all forms of Hg,P ul is the unstirred layer permeability, andP m is the membrane permeability to HgCl2. By fitting this equation to the data we find thatP m =1.3×10−2 cm sec−1. At Cl− concentrations ranging from 1–100mm, diffusion of Hg t through the unstirred layer is rate limiting. At Cl− concentrations ranging from 500–1000mm, the membrane permeability to HgCl2 becomes rate limiting because HgCl2 comprises only about 1% of the total Hg. Under all conditions, chemical reactions among Hg2+, Cl− and/or OH− near the membrane surface play an important role in the transport process. Other important metals, e.g., Zn2+, Cd2+, Ag+ and CH3Hg+, form neutral chloride complexes under physiological conditions. Thus, it is likely that chloride can “facilitate” the diffusion of a variety of metals through lipid bilayer and biological membranes.

On the mechanism of membrane damage by Staphylococcus aureus alpha-toxin.

Abstract: Rabbit or human erythrocytes lysed with Staphylococcus aureus alpha-toxin were solubilized with Triton X-100, and the toxin was subsequently isolated by gel chromatography, sucrose density gradient centrifugation, and reincorporation into liposomes. In the presence of Triton X-100, the toxin exhibited a sedimentation coefficient of 11S and eluted at a position between those of IgG and alpha 2-macroglobulin in gel chromatography. A single polypeptide subunit of 34,000 mol wt was found in SDS PAGE. In the electron microscope, ring-shaped or cylindrical structures were observed, 8.5-10 nm in diameter, harboring central pits or channels 2-3 nm in diameter. An amphiphilic nature of these structures was evident from their capacity to bind lipid and detergent, aggregation in the absence of detergents, and low elutability from biological and artificial membranes through ionic manipulations. In contrast to the membrane-derived form of alpha-toxin, native toxin was a water-soluble, 34,000 mol wt, 3S molecule, devoid of an annular structure. Because studies on the release of radioactive markers from resealed erythrocyte ghosts indicated the presence of circumscribed lesions of approximately 3-nm effective diameter in toxin-treated membranes, the possibility is raised that native alpha-toxin oligomerizes on and in the membrane to form an amphiphilic annular complex that, through its partial embedment within the lipid bilayer, generates a discrete transmembrane channel.

Membrane asymmetry in epithelia: is the tight junction a barrier to diffusion in the plasma membrane?

Abstract: Membrane-bound lectins and some lipid probes are incapable of passing through the tight junction region of epithelial cell membranes. The lectins are immobile on the cell surf ace, but lipid probes diffuse freely in the membrane. The ability of a lipid probe to pass the tight junction is correlated with its ability to ‘flip-flop’ to the inner monolayer of the cell membrane bilayer

Diphtheria toxin fragment forms large pores in phospholipid bilayer membranes

Abstract: The cytotoxic effect of diphtheria toxin requires the entry of its enzymatic A fragment (Mr approximately 21,000) into the cytosol of sensitive cells. We show that the B45 fragment (Mr approximately 24,000) forms, in lipid bilayers, pores that are large enough (diameter greater than or equal to 18 A) to allow the passage of extended fragment A. Pore formation is maximal when the B45-containing side is at low pH (4.7) and the opposite side is at high pH (7.4). These conditions resemble the pH gradient existing across lysosomal membranes. We suggest that fragment A passes through these pores from acidic endocytotic vesicles (lysosomes?) to the cytosol.

Review of reverse osmosis membranes and transport models

Abstract: After a brief introduction to membrane processes in general, and the reverse osmosis process in particular, the structure and properties of membranes and membrane transport theory are described. The mechanism of salt rejection and transport properties of membranes are discussed in detail. Solubility, diffusivity, and permeability of membranes to solutes and solvents are reviewed critically and compared with each other. Special attention is given to two particular types of membranes, cellulose acetate (CA) and aromatic polyamide (AP) membranes, which are often used for water desalination. The major portion of this article is devoted to the review and discussion of membrane transport theory with application to the reverse osmosis and ultrafiltralion processes. It is shown that the solvent flux can be represented reasonably well by linear models such as the solution-diffusion model (Lonsdale, et al., 1965). The contribution of pore flow to the solvent flux is small. The solute flux, however, is not ...

Membrane protein oligomeric structure and transport function.

Abstract: Proteins which traverse membranes tend to have a dimeric structure in which the dimer is arranged asymmetrically across the membrane with the axis of symmetry perpendicular to the membrane plane. This general structure is well suited to the function of transporting nutrients across the cell membrane.

High frequency fusion of plant protoplasts by electric fields.

Abstract: Mesophyll cell protoplasts of Vicia faba were collected by dielectrophoresis in a highly inhomogeneous alternating electric field (sine wave, 5 to 10 V peak-to-peak value, 500 kHz, electrode distance 200 μm). Under these conditions, the cells formed aggregates of two or three on the electrodes or bridges consisting of 4 to 6 protoplasts between the electrodes. This “pearl chain” arrangement of the cells was only stable for the duration of the applied field. By the additional application of a high single field pulse (square wave, 15 V, 50 μs), it was possible to induce cell fusion within the aggregates or bridges. This electrically stimulated fusion of cells proceeded at room temperature and under physiological pH-conditions, without the use of chemical reagents, and gave a high yield. Smaller fused aggregates formed spheres within a few minutes. During the dielectrophoretically induced adhesion of the protoplasts to one another, the field strength must be chosen such that dielectric breakdown of the membrane is avoided, but at the same time, the strength of the subsequently applied single field pulse must be high enough to induce dielectric breakdown at the sites of contact between the protoplast membranes. From these results, one can conclude that in addition to close contact between membranes, the prerequisite for electrically stimulated cell fusion is dielectric breakdown which leads to changes in the membrane conductance, permeability, and probably fluidity.

In situ electrophoresis of membrane components

Abstract: During the past decade, the realization that cell membrane constituents may exist in a fluid matrix, each constituent capable of exhibiting rapid rotational and translational motions within the membrane (20, 70), has not only reshaped our concepts about the structure and function of the cell membrane, but also has brought about a number of new problems in membrane biology. The interactions among membrane components (25), lateral mobilities of lipids and proteins (20, 49), and mechanisms for stabilizing and modulating membrane topography (19, 47, 48) are some of the subjects currently under extensive investigation. One ap­ proach to these problems is to perturb experimentally the steady state topography of the membrane components and then examine the struct­ ural and functional consequences of such a perturbation. In situ elec­ trophoresis is one of the newly developed methods in this experimental approach. It is based on the idea that charged membrane components, if free to move laterally in the plane of the membrane, should undergo redistribution in response to an electric field applied along the mem­ brane surface. This "lateral electrophoresis" in cell membranes was first suggested by Jaffe as a possible cellular mechanism underlying the effects of biogenic electric field in developing systems (31). The study of in situ electrophoresis of membrane components is still in a state of infancy. Only half a dozen publications have appeared since the first reports in 1977 (31, 61). Thus, the purpose of the present paper is not simply to review this relatively scarce and recent literature but to provide a concise theoretical framework for the use of in situ electrophoresis, to summarize lessons learned in the experimental stud­ ies, and to point out possible strategies for the application of this

Selective labeling of the hydrophobic core of membranes with 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine, a carbene-generating reagent.

Abstract: The synthesis of a new photoactivatable probe, 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine ([125I]-TID), with a high specific radioactivity (10 Ci mmol-1) is described. It was tested as a probe for the hydrophobic core of membranes. TID partitions strongly in favor of the lipid phase of membranes, and the photogenerated carbene labels intrinsic membrane proteins in a highly selective manner. This conclusion was reached from the distribution of radioactivity among the proteins of [125I]TID-labeled human erythrocyte membranes. By far the most heavily labeled protein is band 3 [nomenclature of Fairbanks, G., Steck, T. L., & Wallach, F. G. H. (1971) Biochemistry 10, 2606-2617] while the labeling of glycophorin is approximately 5 times less than that of band 3. There is little or no labeling of known extrinsic proteins.

Fluorescence quenching in model membranes. 3. Relationship between calcium adenosinetriphosphatase enzyme activity and the affinity of the protein for phosphatidylcholines with different acyl chain characteristics.

Abstract: The dependence of function and lipid binding affinity of an integral transport protein on the fatty acyl chain characteristics of a membrane-forming phospholipid have been determined. When a newly developed fluorescence quenching technique [London, E., & Feigenson, G. W. (1981) Biochemistry (first paper of three in this issue); London, E., & Feigenson, G. W. (1981) Biochemistry (preceding paper in this issue)] is used for examining lipid-protein interactions in membranes, the Ca2+ ATPase from rabbit sarcoplasmic reticulum is found to bind with equal affinity a large variety of phosphatidylcholines used to reconstitute the protein into enzymatically active vesicles, regardless of fatty acyl chain length or details of unsaturation. In parallel with the lipid binding studies, we have measured the sensitivity of the catalytic activity of the Ca2+ ATPase to the fatty acyl chain characteristics of the phosphatidylcholine membranes in which the enzyme was reconstituted. The enzyme appears to be sensitive only to the effective fatty acyl chain length, which determines the thickness of the bilayer in which the protein is inserted and displays little sensitivity to such details of unsaturation as degree, position, and isomeric type. Both ATP hydrolyzing and Ca2+ transporting activities of the enzyme were similarly affected by bilayer thickness, and maximum activity was observed in membranes of intermediate thickness. These observations are reconciled in a number of possible models for the manner in which this integral protein interacts with membranes of varying thickness. A freeze-thaw method was used to reconstitute the Ca2+ ATPase, and the vesicles so obtained have been characterized by gel permeation chromatography, density gradient centrifugation, and electron microscopy, (thin section). Convenient methods are described for (a) rapidly separating reconstituted Ca2+ ATPase from unincorporated protein simultaneously in a large number of small samples, giving good recovery of fractionated vesicles without significant dilution, and (b) measuring leakiness to Ca2+ of reconstituted vesicles. Additionally, the gel and liquid-crystal phase transition temperature and bilayer thickness have been determined respectively by differential thermal analysis and low-angle X-ray diffraction for some of the synthetic phosphatidylcholines, which range in chain length from 12 to 24 carbon atoms.

A relationship between alcohol intoxication and the disordering of brain membranes by a series of short-chain alcohols.

Abstract: This study has established a correlation between the hypnotic potencies of aliphatic alcohols and their abilities to disrupt the structure of neuronal membranes in vitro. The hypnotic potency was determined in mice from the ED50 for loss of righting reflex. The alcohol-induced perturbation of mouse brain synaptosomal plasma membranes was measured by a sensitive electron paramagnetic resonance technique. The membrane disordering potency was determined from the slope of the concentration-dependent decrease in order parameter observed for each alcohol. Significant reductions in the order parameter were observed at nerve blocking concentrations. The following alcohols were investigated: ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, 2-methyl-1-propanol, 2-methyl-2-propanol, 1-pentanol, 2-pentanol, 3-methyl-1-butanol, 1-hexanol and 1-octanol. The disordering potency of each alcohol was closely related to its membrane solubility, based on published oil/water partition coefficients. Structural disorganization resulting from the incorporation of alcohols into neuronal membranes may be an integral step in the mechanism of alcohol intoxication. For a given degree of membrane disorder, intramembrane alcohol concentrations and intramembrane alcohol volumes were estimated from published partitioning and molecular volume data and compared for constancy. The data did not favor either the intramembrane drug concentration or the intramembrane drug volume as a more effectual determinant of disordering potency.

Xenon NMR: Chemical shifts of a general anesthetic in common solvents, proteins, and membranes

Abstract: The rare gas xenon contains two NMR-sensitive isotopes in high natural abundance. The nuclide 129Xe has a spin of 1/2: 131Xe is quadrupolar with a spin of 3/2. The complementary NMR characteristics of these nuclei provide a unique opportunity for probing their environment. The method is widely applicable because xenon interacts with a useful range of condensed phases including pure liquids, protein solutions, and suspensions of lipid and biological membranes. Although xenon is chemically inert, it does interact with living systems; it is an effective general anesthetic. We have found that the range of chemical shifts of 129Xe dissolved in common solvents is ca. 200 ppm, which is 30 times larger than that found for 13C in methane dissolved in various solvents. Resonances were also observed for 131Xe in some systems; they were broader and exhibited much greater relaxation rates than did 129Xe. The use of 129Xe NMR as a probe of biological systems was investigated. Spectra were obtained from solutions of myoglobin, from suspensions of various lipid bilayers, and from suspensions of the membranes of erythrocytes and of the acetylcholine receptor-rich membranes of Torpedo californica. These systems exhibited a smaller range of chemical shifts. In most cases there was evidence of a fast exchange of xenon between the aqueous and organic environments, but the exchange was slow in suspensions of dimyristoyl lecithin vesicles.

Early and late functions associated with the Golgi apparatus reside in distinct compartments.

Abstract: Enzymes that catalyze the two successive stages of Golgi-associated processing of asparagine-linked oligosaccharides distributed differently when membranes from Chinese hamster ovary cells were centrifuged in a sucrose density gradient. A mannosidase that removes only outer, alpha-1,2-linked mannose residues from the precursor oligosaccharides of the vesicular stomatitis viral G protein (to yield a "trimmed" oligosaccharide core) was separated from enzymes (galactosyl- and sialyltransferases) that act in the later, terminal stage of glycosylation. Freshly acylated G protein with newly trimmed oligosaccharides banded in the distribution of early-acting membranes, defined by the mannosidase, whereas G protein pulse-labeled with [3H]galactose distributed in the profile of the late-acting membranes. G protein present in the early-acting membranes in crude fractions could be terminally glycosylated by incubation with exogenous Golgi membranes in vitro; G protein lost its ability to be processed in vitro as it appeared to enter the late-acting membranes in vivo. These experiments reveal the existence of two distinct compartments through which intracellularly transported proteins such as G pass in sequence as Golgi-associated processes are carried out. It is likely that these compartments consist of cisternae on the cis and trans sides of the Golgi stack.

Cells with Manipulated Functions: New Perspectives for Cell Biology, Medicine, and Technology

Abstract: Exposure to electrical fields can reversibly increase the electrical conductivity and permeability of a cell membrane, which regulates and directs the exchange of materials and information between the cell and its environment. If cell membranes (or artificial lipid membranes) are exposed to a field pulse of high intensity and short duration (ns to μs), local electrical breakdown occurs in them. This electrical breakdown is associated with a large permeability change in the membrane, which is such that substances or particles (up to the size of genes) which cannot normally permeate through the membrane, are able to traverse the membrane into the cell. The original properties of the membrane are restored within μs to min, depending on the experimental conditions and the membrane properties. Electrical breakdown in the zone of contact between the membranes of cells (or lipid vesicles), which have been made to adhere to each other by the action of weak inhomogeneous alternating electrical fields, leads to fusion of these cells with formation of a single cell having new functional characteristics. The electrical fusion method is very mild, and the yield of fused cells is high. The electrically induced fusion and entrapment of membrane-impermeable substances and genes in cells provide a new tool for the productions of a wide range of cells with manipulated functions, which could be used (or are being used) for the solution of a number of problems in cell biology, medicine and technology. The application of electrical membrane breakdown to clinical diagnostics, the development of cellular carrier systems for the selective transport of drugs to a site of action within the organism and the potential applications of electrically induced fusion for breeding salt-tolerant crop plants for converting solar energy into ethanol, for synthesizing natural materials and manipulating genes, are described.