Showing papers on "Membrane published in 1977"

Electrostatic Potentials at Membrane-Solution Interfaces

Abstract: Publisher Summary This chapter discusses electrostatic potentials at membrane solution interfaces. Lipids in the membranes of all cells and subcellular organelles are arranged in the form of a bilayer with the hydrocarbon tails sequestered away from the water and the polar head groups exposed to the aqueous environment. About 10%–20% of the lipids in the membranes of many cells and organelles bear a net negative charge, whereas positively charged lipids are extremely rare. The concentration of monovalent cations at the surface of the bilayer will be an order of magnitude higher than the concentration of these ions in the bulk aqueous phase. The surface potential produced by charged lipids is dependent on the salt concentration in the bulk aqueous phase and a seminal paper. The absence of proteins, polysaccharides, and other macromolecules present in biological membranes can be considered an advantage to test how well the theory of the diffuse double layer describes the electrostatic potential produced by charges on lipids. The chapter describes hydrophobic adsorption of charged molecules to bilayer membranes, the electrostatic potential produced by molecular dipoles at membrane-solution interfaces, and the electrostatic boundary potential produced by charges located in the interior of the membrane, a few angstroms from the interface. Few examples of the possible biological significance of these electrostatic surface potentials are also described.

Polymyxin and related peptide antibiotics.

Abstract: The general mechanism for the antibiotic activities of the polymyxins and octapeptins has been elucidated by research using a broad range of experimental techniques. However, this phenomenon has not been described in detailed molecular terms, and this must be one of the major goals for future research in this area. Since 1947, when polymyxin was first isolated, there have been tremendous advances in our knowledge of membrane structure. The application of biophysical technology such as NMR, ESR, fluorescence spectroscopy, differential scanning calorimetry, and electron microscopy has been particularly valuable for studying model and biological membrane structures. It is these techniques which will provide a detailed molecular mechanism for the effects of these peptide antibiotics on membrane structure. In addition, the large number of antibiotic derivatives available should be exploited more extensively for structure-function correlations. The ultimate goal is to correlate the biological properties of these peptides with their effects on the physical properties of membranes and to rationalize these events in terms of lipid-peptide interactions.

Formation and resealing of pores of controlled sizes in human erythrocyte membrane

Abstract: APPLICATION of an electric pulse, at field intensities of a few kV cm−1 and of duration in the µs range, to an isotonic suspension of erythrocytes is known to cause haemolysis of the red cells1–4. Studies from different laboratories suggest that the haemolysis is due to the field-induced transmembrane potential1,3,4. Our recent experiments5 indicate that once the transmembrane potential reaches a threshold of approximately 1 V, which corresponds to an applied field of 2.2 kV cm−1, the erythrocyte membrane becomes leaky to normally impermeant ions or molecules. The permeation of solutes leads to the swelling and eventual lysis of the red cells. This type of haemolysis is known as colloid osmotic haemolysis6,7. The voltage-induced permeability change is consistent with the formation of pores in the membrane. We show here that the size of these pores can be varied in a controlled manner, and that the leaky membrane can be resealed while the haemolysis is prevented. Foreign molecules have successfully been incorporated into the resealed, but otherwise intact, erythrocytes.

Synchronised transmembrane insertion and glycosylation of a nascent membrane protein

Abstract: Studies of the synthesis and incorporation of the vesicular stomatitis virus glycoprotein into membranes in a synchronised cell-free system demonstrate a tight coupling between polypeptide synthesis and membrane insertion, as a result of which the nascent chain crosses the membrane. The studies reveal a surprisingly precise sequence by which the nascent chain of this membrane glycoprotein is glycosylated in two steps. These findings have important implications for the mechanisms of membrane assembly.

Drug tolerance in biomembranes: a spin label study of the effects of ethanol

Abstract: Ethanol in vitro increased the fluidity of spin-labeled membranes from normal mice. Membranes from mice that had been subjected to long-term ethanol treatment were relatively resistant to this fluidizing effect. The data suggest that the membranes themselves had adapted to the drug, a novel form of drug tolerance.

Effects of low concentrations of ethanol on the fluidity of spin-labeled erythrocyte and brain membranes.

Abstract: The effects of ethanol on membrane fluidity at 37° have been assessed by a sensitive electron paramagnetic resonance technique. Erythrocyte and brain membranes from DBA/2J mice were spin-labeled with 5-doxylstearic acid ( N -oxyl-49,49-dimethyloxazolidine derivative of 5-ketostearic acid). The molecular motion of the spin label was measured from the EPR spectrum by determining the order parameter S , an index of membrane fluidity. The fluidity of both erythrocyte and synaptosomal membranes was greater than that of myelin but less than that of mitochondrial membranes. The addition of low concentrations (0.02 or 0.04 M) of ethanol in vitro increased fluidity in erythrocyte, mitochondrial, and synaptosomal membranes. This fluidizing effect of ethanol was dose-related up to 0.35 M in all the membranes except myelin. These data suggest that nonlethal concentrations of ethanol may increase membrane fluidity in vivo .

Abnormalities of cell-membrane fluidity in the pathogenesis of disease.

Abstract: Early in this century, colloid chemists debated about whether cells were bounded by a surface membrane. Today, they debate about the organization of molecules within that membrane. The research involved has led to two images of the cell membrane — as a sea of lipid with islands of protein and as a matrix of protein with lakes of lipid. Both concepts suggest that zones of fluid lipid within the membrane form the environment for membrane proteins. Much of this lipid is in the form of a bilamellar leaflet with hydrophilic portions that face the aqueous environment on either side and . . .

Multicomponent membranes for gas separations

Abstract: Multicomponent membranes for gas separation are disclosed which are effective for separating at least one gas from gaseous mixtures by permeation wherein the multicomponent membranes are comprised of a coating in occluding contact with a porous separation membrane. The multicomponent membranes advantageously exhibit less resistance to permeate gas flow for the at least one gas than for at least one remaining gas in the gaseous mixture. For at least one pair of gases the separation factor of the multicomponent membrane is significantly greater than the determined intrinsic separation factor of the material of the coating. Processes and apparatus for gas separation utilizing the multicomponent membranes are also disclosed.

Large-scale purification of the acetylcholine-receptor protein in its membrane-bound and detergent-extracted forms from Torpedo marmorata electric organ.

Abstract: A method is described for the large-scale purification of membrane fragments very rich in acetylcholine (nicotinic) receptor from the electric organ of Torpedo marmorata. The preparations of purified membrane fragments have a specific activity of more than 4000 nmol α-toxin binding sites/g protein and give only four main polypeptide bands by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Observations by electron microscopy show that the purified preparation of receptor-rich membrane fragments is composed of only one class of membrane fragments covered with 8-nm rosettes identified as acetylcholine receptor molecules. This preparation is used as a starting material for the detergent solubilization and the large-scale purification of the acetylcholine receptor protein, without using affinity chromatography. A sucrose gradient centrifugation of a Triton X-100 extract of receptor-rich membranes done in the presence of 2-mercaptoethanol yields large quantities of receptor protein in a homogeneous form as indicated by polyacrylamide gel electrophoresis, isoelectric focussing and electron microscopy. Polyacrylamide gel electrophoresis of the purified protein in the presence of sodium dodecylsulfate reveals three bands of apparent molecular weights 40000 ± 1000, 50000 ± 2000 and 66000 ± 2000, the two heaviest ones being present in significantly lower amounts than the 40000-Mr one. The comparison of several samples of purified protein with different specific activities reveals a striking variability of the ratios of the 40000-Mr band to the 50000 and 66000-Mr ones.

A study of the ion selectivity and the kinetic properties of the calcium dependent slow inward current in mammalian cardiac muscle.

Abstract: 1. A voltage-clamp method combining a single surcose gap and two intracellular micro-electrodes was used to measure membrane currents in ventricullar myocardial fibres. 2. The adequacy of the voltage-clamp method is demonstrated by comparing the total current, It, across the gap with the voltage difference, delta V, between the two intracellular micro-electrodes, i.e. another independent way of measuring membrane currents. With both current measurements the slow inward current, Is, shows the same voltage- and time-dependences. 3. The sensitivity of the slow inward current to variation in external Ca and Na concentrations was investigated systematically. The reversal potential of the slow inward current was sensitive to variation of both ion species. 4. From the reversal potential measurements relative permeabilities of the conductance channels of the slow inward current were estimated as PCa/PNa approximately 1/0-01 and PCa/PK approximately 1/0-01 by means of the constant field equation. 5. The activation and inactivation kinetics of the slow inward current were explored in detail and related to the plateau of the action potential.

Evidence for decreased amounts of two major glycoproteins

Abstract: A B S T R A C T Platelets from patients with Glanzmann's thrombasthenia have a distinct molecular alteration of the plasma membrane surface, namely decreased amounts of a major glycoprotein designated as Ilb (apparent mol wt 142,000). To identify other possible surface defects of thrombasthenic platelets, we labeled the membrane polypeptides of normal and thrombasthenic platelets by two different techniques: lactoperoxidase-catalyzed iodination and galactose oxidase oxidation, followed by reduction with tritiated sodium borohydride. Labeling patterns were determined after the polypeptides were separated by twodimensional polyacrylamide gel electrophoresis. Before the second dimension was run, platelet samples were incubated with a reducing agent, 8-mercaptoethanol, to cleave the disulfide bonds of certain glycoproteins; the resulting changes in electrophoretic mobility permitted better resolution of individual molecules. Comparison of the labeled polypeptides of normal and thrombasthenic samples after reduction indicated decreased labeling of two major glycoproteins in thrombasthenic platelets: IIb and III (apparent mol wt 114,000). The relative proportions of radioactivity incorporated by these polypeptides were about 60 and 80% less than control values, respectively. With either Coomassie Blue or periodic acidSchiff's reagent, glycoprotein III stained much less intensely in thrombasthenic compared to normal samples, indicating that the observed labeling deficit was caused by a decreased concentration of the molecule rather than steric inaccessibility on the membrane surface. Analysis of normal plasma membranes by afDr. Phillips is a recipient of a Research Career Development Award, HL-0800, from the National Heart and Lung

Interaction between the third complement protein and cell surface macromolecules.

Abstract: The activated form of the third complement protein, C3b, forms a stable complex with components of plasma membranes and particulate entities such as zymosan. The complex resists the action of detergents and protein denaturants as well as extremes of temperature, salt concentration, and pH. It can, however, be broken by exposure to hydroxylamine or by ammonolysis followed by incubation with sodium dodecyl sulfate. Thus, the complex appears to result from a hydrophobic interaction as well as a bond susceptible to nucleophilic attack.

Current-voltage curve of sodium channels and concentration dependence of sodium permeability in frog skin.

Abstract: 1. The inward facing membranes of in vitro frog skin epithelium were depolarized with solutions of high K concentration. The electrical properties of the epithelium are then expected to be governed by the outward facing, Na-selective membrane.2. In this state, the transepithelial voltage (V) was clamped to zero and step-changes of Na activity in the outer solution ((Na)(o)) were performed with a fast-flow chamber at constant ionic strength, while the short-circuit current was recorded.3. At pre-selected times after a step-change of (Na)(o) the current response (I) to a fast voltage staircase was recorded. This procedure was repeated after blocking the Na channels with amiloride to obtain the current-voltage curve of transmembrane and paracellular shunt pathways. The current-voltage curve of the Na channels was computed by subtracting the shunt current from the total current.4. The instantaneous I(Na)-V curve thus obtained at a given (Na)(o) could easily be fitted with the constant field equation in the range between -50 and zero mV. This fit yielded approximate estimates of P(Na), the Na- permeability of the Na-selective membrane (at this (Na)(o)) and the cellular Na activity, (Na)(c). As residual properties of the serosal membrane were ignored the computed values are expected to underestimate the true ones.5. At constant (Na)(c), the steady-state value of 1/P(Na) increases linearly with (Na)(o). Error analysis and the effect of drugs show that the dependence is not due to the residual properties of the inward facing membranes but reflects the true behaviour of P(Na).6. The steady-state P(Na) at a given (Na)(o) is smaller than the transient P(Na) observed right after a stepwise increase of (Na)(o) to this value. The time constant of P(Na)-relaxation is in the order of seconds.7. In conclusion, Na transport through open Na-selective channels of the outward facing membrane of the stratum granulosum cells can be described as an electrodiffusion process which as such does not saturate with increasing (Na)(o). However, when added to the outer border of the membrane Na causes a decrease of P(Na) within several seconds. It is considered that binding of Na results in closure of Na channels.

Release of ovoperoxidase from sea urchin eggs hardens the fertilization membrane with tyrosine crosslinks.

Abstract: One feature of fertilization is the alteration of the vitelline layer, by components released from the egg, to produce an elevated, covalently crosslinked, hard, insoluble, fertilization membrane. The following evidence indicates that crosslinking and hardening are caused by the production of diand trityrosyl residues, by oxidation of protein-bound tyrosyl residues in the presence of a peroxidase. Hardening of the fertilization membrane, as evidenced by its loss of solubility in 50 mM dithiothreitol, is inhibited by compounds known to inhibit many peroxidases. A peroxidase, here called the ovoperoxidase, is released from eggs at fertilization. This enzyme is inhibited by the same compounds that inhibit hardening and at similar concentrations. Inhibitors of the ovoperoxidase and the hardening reaction include KCN, 3-amino-1,2,4-triazole, NaN(3), phenylhydrazine, K(4)Fe(CN)(6), sodium sulfite, and glycine ethyl ester. In addition, tyramine and N-acetyltyrosine both inhibit hardening, but O-methyltyrosine does not. Dityrosyl and trityrosyl residues are found in acid hydrolysates of isolated, hardened fertilization membranes. These residues have been identified by cellulose phosphate column chromatography, thin-layer chromatography, and amino acid analysis. The amino acid data have been used to estimate that there is one dityrosine crosslink per 55,000 daltons of protein. We suggest that, by catalyzing the crosslinking of tyrosyl residues, the ovoperoxidase leads to the production of a hard fertilization membrane that blocks the entry of additional sperm. Because peroxidases are spermicidal, a secondary function of the enzyme could be to kill sperm in the vicinity of the fertilized egg.

Conversion of lamellar body membranes into tubular myelin in alveoli of fetal rat lungs.

Abstract: Fluid-filled lumina of fetal rat lungs contain lamellar bodies (LBs) as well as tubular myelin (TM), both of which are thought to be stores of phospholipid-rich pulmonary surfactant. The alveolar epithelium is believed to secrete LBs, but neither the origin nor the mechanism of TM formation is entirely certain. The main objective of this study was to determine the relationship between secreted LBs and TM and to define membrane phenomena which occur during TM formation. I examined lung tissues of 20-21 day-old fetuses (day 22 = term) using transmission and high voltage transmission electron microscopy and cytochemistry. My findings indicate that secreted LBs, identified by the presence of an acid-phosphatase reactive core, are the precursor of TM. Secreted LBs are highly organized structures which contain structurally specialized areas, one of which is a "mini-lattice" structure similar to TM. During TM formation, fuzzes or 8.0-nm diameter particles appear on transition membranes, although LB membranes appear to lack both structures. Similar particles are present on TM membranes and are generally associated with membrane intersections. My results provide evidence that TM is formed from LBs within the alveolar lumen by mechanisms which may be complex.

Electrophoresis along cell membranes

Abstract: Bioelectric fields may segregate charged components floating in the plasma membranes of cells by a process of electrophoresis along the membrane. Molecules in cell membranes may be sorted to different portions of the cell surface by such electrical gradients. We present here a theory to support this hypothesis.

Rapid isolation of plasma membranes in high yield from cultured fibroblasts.

Abstract: 1. A method was developed which allows the rapid preparation of pure plasma membranes in high yield from cultured fibroblasts. 2. Cells are lysed in hypo-osmotic borate/EDTA and, after differential centrifugation, the membranes collected by centrifugation on a sucrose barrier. 3. Electron microscopy of the isolated material shows large membrane vesicles essentially free from contaminating organelles. 4. There is no detectable activity of the endoplasmic-reticulum enzyme marker, NADH2--lipoamide oxidoreductase (EC 1.6.4.3), and that of succinate dehydrogenase (EC 1.3.99.1), a marker for mitochondria, is substantially decreased. Chemical compositions are in good agreement with previous observations. 5. This study confirms the usefulness of applied isotopic markers for isolating plasma membranes.

Resonance Raman studies of the purple membrane

Abstract: The individual resonance Raman spectra of the PM568 and M412 forms of light-adapted purple membrane from Halobacterium halobium have been measured using the newly developed flow technique. For comparison purposes, the Raman spectra of the model chromophores, all-trans- and 13-cis retinal n-butylamine, both as protonated and unprotonated Schiff bases, have also been obtained. In agreement with previous work, the Raman data indicate that the retinal chromophore is linked to the purple membrane protein via a protonated. Schiff base in the case of the PM568 and an unprotonated Schiff base for the M412 form. The basic mechanism for color regulation in both forms appears to be electron delocalization. The spectral features of the two forms are different from each other and different from the model compound spectra.

Molecular events during membrane fusion. A study of exocytosis in rat peritoneal mast cells.

Abstract: We have used thin section and freeze-fracture electron microscopy to study membrane changes occurring during exocytosis in rat peritoneal mast cells. By labeling degranulating mast cells with ferritin-conjugated lectins and anti-immunoglobulin antibodies, we demonstrate that these ligands do not bind to areas of plasma membrane or granule membrane which have fused with, or are interacting with, granule membrane. Moreover, intramembrane particles are also largely absent from both protoplasmic and external fracture faces of plasma and granule membranes in regions where these membranes appear to be interacting. Both the externally applied ligands and intramembrane particles are sometimes concentrated at the edges of fusion sites. The results indicate that membrane proteins are displaced laterally into adjacent membrane regions before the fusion process and that fusion occurs between protein-depleted lipid bilayers. The finding of protein-depleted blebs in regions of plasma and granule membrane interaction raises the interesting possibility that blebbing may be a process for exposing the granule contents to the extracellular space and for the elimination of excess lipid while conserving membrane proteins.

Sites and specificity of the reaction of bipyridylium compounds with anaerobic respiratory enzymes of Escherichia coli. Effects of permeability barriers imposed by the cytoplasmic membrane

Abstract: The ability of the oxidized and singly reduced species of several bipyridylium cations to cross the cytoplasmic membrane of Escherichia coli was studied to locate the sites of reaction of the dyes with anaerobic respiratory enzymes Benzyl Viologen radical crossed the membrane rapidly, whereas the oxidized species did not The oxidized or radical species of Methyl Viologen, Morfamquat or Diquat did not rapidly cross the membrane It was also shown that the dithionite anion does not cross the cytoplasmic membrane of E coli Diquat radical donates electrons to the nitrate reductase pathway at the periplasmic aspect of the membrane, whereas Benzyl Viologen radical reacted directly with nitrate reductase itself (EC 17994) at the cytoplasmic aspect of the membrane Thus the pathway of electron transfer in the nitrate reductase pathway is transmembranous Formate hydrogenlyase (EC 1212) and an uncharacterized nitrite reductase activity react with bipyridylium dyes at the periplasmic aspect of the membrane Fumarate reductase (succinate dehydrogenase; EC 13991) reacts with bipyridylium radicals, and formate dehydrogenase (cytochrome) (EC 1221) with ferricyanide, at the cytoplasmic aspect of the membrane The differing charge and membrane permeation of oxidized and radical species of bipyridylium dyes greatly complicate their use as potentiometric mediators in suspensions of cells or membrane vesicles

Release of spectrin-free vesicles from human erythrocytes during ATP depletion: 1. characterization of spectrin-free vesicles

Abstract: Human erythrocytes incubated without glucose at 37 degrees C (in vitro aging) release spectrin-free vesicles after 12 or more hours. The release of vesicles is dependent upon ATP depletion. If the endogenous level of ATP is maintained, vesicle release is completely inhibited up to 54 h. Vesicle release is independent of hemolysis because in vitro aged cells and cells that maintain their ATP levels lose identical amounts of hemoglobin up to 45 h. 93 percent of all membrane particles released constitute a uniform population of spheres with a diameter of 185 +/- 23nm. These vesicles are of slightly varying densities due to varying contents of hemoglobin. Vesicles contain half the amount of membrane protein that is found in intact membranes when referred to the content of phospholipids phosphorus. This is primarily due to the absence of spectrin. However, their content of protein component III, glycophorin, and cholesterol remains the same as in intact membranes. Thus, the major integral membrane proteins are present in vesicles in similar quantities were surface area as in cells except for the enzyme acetylcholinesterase that is enriched up to twofold. The phospholipids composition of these vesicles is representative of the intact membrane except that the amount of phosphatidic acid is 10-fold higher and the amount of phosphatidylethanolamine is slightly lower than in erythrocytes. These results suggest a selective release of membrane domains that lack peripheral membrane proteins and are enriched in acetylcholinesterase. This release of spectrin-free vesicles from cells aged in vitro could represent an acceleration of the physiological aging process.