Showing papers on "Membrane published in 1976"

Role of ubiquinone in the mitochondrial generation of hydrogen peroxide

Abstract: Antimycin-inhibited bovine heart submitochondrial particles generate O2- and H2O2 with succinate as electron donor. H2O2 generation involves the action of the mitochondrial superoxide dismutase, in accordance with the McCord & Fridovich [(1969) j. biol. Chem. 244, 6049-6055] reaction mechanism. Removal of ubiquinone by acetone treatment decreases the ability of mitochondrial preparations to generate O2- and H2O2, whereas supplementation of the depleted membranes with ubiquinone enhances the peroxide-generating activity in the reconstituted membranes. Addition of superoxide dismutase to ubiquinone-reconstituted membranes is essential in order to obtain maximal rates of H2O2 generation since the acetone treatment of the membranes apparently inactivates (or removes) the mitochondrial superoxide dismutase. Parallel measurements of H2O2 production, succinate dehydrogenase and succinate-cytochrome c reductase activities show that peroxide generation by ubiquinone-supplemented membranes is a monotonous function of the reducible ubiquinone content, whereas the other two measured activities reach saturation at relatively low concentrations of reducible quinone. Alkaline treatment of submitochondrial particles causes a significant decrease in succinate dehydrogenase activity and succinate-dependent H2O2 production, which contrasts with the increase of peroxide production by the same particles with NADH as electron donor. Solubilized succinate dehydrogenase generates H2O2 at a much lower rate than the parent submitochondrial particles. It is postulated that ubisemiquinone (and ubiquinol) are chiefly responsible for the succinate-dependent peroxide production by the mitochondrial inner membrane.

Fluorescence Depolarization Studies of Phase Transitions and Fluidity in Phospholipid Bilayers. 2. Two-Component Phosphatidylcholine Liposomes

Abstract: The fluorescence depolarization associated with the hydrophobic fluorescent probe 1,6-diphenyl-1,3,5-hexatriene is used to monitor the changes in fluidity accompanying the gel-liquid crystalline phase transition in synthetic phosphatidycholine dispersions. The parameters of the phase transition are determined for both large, multilamellar liposomes and small, single-lamellar vesicles. These parameters are compared with those obtained using other techniques. In addition, the data are interpreted in terms of two limiting molecular models, which in turn offer insight into the structural differences between multilammelar liposomes and small vesicles.

Sodium/proton antiport in brush-border-membrane vesicles isolated from rat small intestine and kidney.

Abstract: Studies on proton and Na+ transport by isolated intestinal and renal brush-border-membrane vesicles were carried out to test for the presence of an Na+/H+-exchange system. Proton transport was evaluated as proton transfer from the intravesicular space to the incubation medium by monitoring pH changes in the membrane suspension induced by sudden addition of cations. Na+ transport was determined as Na+ uptake into the vesicles by filtration technique. A sudden addition of sodium salts (but not choline) to the membrane suspension provokes an acidification of the incubation medium which is abolished by the addition of 0.5% Triton X-100. Pretreatment of the membranes with Triton X-100 prevents the acidification. The acidification is also not observed if the [K+] and proton conductance of the membranes have been increased by the simultaneous addition of valinomycin and carbonyl cyanide p-trifluoromethoxyphenylhydrazone to the K+-rich incubation medium. Either valinomycin or carbonyl cyanide p-trifluoromethoxyphenylhydrazone when added alone do not alter the response of the membranes to the addition of Na+. Na+ uptake by brush-border microvilli is enhanced in the presence of a proton gradient directed from the intravesicular space to the incubation medium. Under these conditions a transient accumulation of Na+ inside the vesicles is observed. It is concluded that intestinal and renal brush-border membranes contain a NA+/H+ antiport system which catalyses an electroneutral exchange of Na+ against protons and consequently can produce a proton gradient in the presence of a concentration difference for Na+. This system might be involved in the active proton secretion of the small intestine and the proximal tubule of the kidney.

Measurement of forces between lecithin bilayers.

Abstract: CELL fusion1 and vesicle-membrane2 or vesicle-synapse3 fusion are currently thought to involve distance-dependent interactions between membranes. We have devised a general method for measuring such forces between planar phospho-lipid membranes, and report here results for the specific case of egg lecithin (phosphatidyl choline) bilayers in water.

Uptake and retention of metals by cell walls of Bacillus subtilis.

Abstract: Isolated walls of Bacillus subtilis Marburg, prepared in a manner which avoided metal contamination other than by the growth medium, were incubated in dilute metal solutions, separated by membrane filtration (0.22 mum), and monitored by atomic absorption to give uptake data for 18 metals. Substantial amounts of Mg2+, Fe3+, Cu2+, Na+, and K+ (amounts which were often visible as Au3+, and Ni2+ (the higher atomic-numbered elements also visible as electron scattering), and small amounts of Hg2+, Sr2+, Pb2+, and Ag+ were taken into the wall. Some (Li+, Ba2+, Co2+, and Al3+) were not absorbed. Most metals which had atomic numbers greater than 11 and which could be detected by electron microscopy appeared to diffusely stain thin sections of the wall. Magnesium, on the other hand, partitioned into the central region, and these sections of walls resisted ruthenium red staining, which was not true for the other metals. Areas of the walls also acted as nucleation sites for the growth of microscopic elemental gold crystals when incubated in solutions of auric chloride. Retention or displacement of the metals was estimated by a "chromatographic" method using the walls cross-linked by the carbodiimide reaction to adipic hydrazide agarose beads (which did not take up metal but reduced the metal binding capacity of the walls by ca. 1%) packed in a column. When a series of 12 metal solutions was passed through the column, it became evident that Mg2+, Ca2+, Fe3+, and Ni2+ were strongly bound to the walls and could be detected by both atomic absorption and by their electron-scattering power in thin sections, qhereas the other metals were fisplaced or replaced. Partial lysozyme digestion of the walls (causing a 28% loss of a [3H]diaminopimelic acid label) greatly diminished the Mg2+ retention but not that of Ca2+, Fe3+, or Ni2+, indicating that there are select sites for various cations.

Reconstitution in planar lipid bilayers of a voltage-dependent anion-selective channel obtained from paramecium mitochondria.

Abstract: We have incorporated into planar lipid bilayer membranes a voltage-dependent, anion-selective channel (VDAC) obtained fromParamecium aurelia. VDAC-containing membranes have the following properties: (1) The steady-state conductance of a many-channel membrane is maximal when the transmembrane potential is zero and decreases as a steep function of both positive and negative voltage. (2) The fraction of time that an individual channel stays open is strongly voltage dependent in a manner that parallels the voltage dependence of a many-channel membrane. (3) The conductance of the open channel is about 500 pmho in 0.1 to 1.0m salt solutions and is ohmic. (4) The channel is about 7 times more permeable to Cl− than to K+ and is impermeable to Ca++. The procedure for obtaining VDAC and the properties of the channel are highly reproducible.

Adaptation of membrane lipids to alcohols.

Abstract: The effects of alcohols of different chain lengths on the fatty acid composition of Escherichia coli K-12 have been examined. My results indicate that these cells radically change their fatty acid composition when grown in the presence of alcohols. These changes represent an adaptive membrane alteration compensating for the direct physicochemical interaction of alcohols with the membrane. Similar adaptive responses of membrane lipids are proposed as a possible biochemical basis for tolerance to alcohol and related drugs.

Water and Nonelectrolyte Permeability of Lipid Bilayer Membranes

Abstract: Both the permeability coefficients (Pd's) through lipid bilayer membranes of varying composition (lecithin [L], lecithin:cholesterol [LC], and spingomyelin:cholesterol [SC]) and the n-hexadecane:water partition coefficients (Knc's) of H2O and seven nonelectrolytes (1,6 hexanediol, 1,4 butanediol, n-butyramide, isobutyramide, acetamide, formamide, and urea) were measured. For a given membrane compositiin, Pd/DKnc (where D is the diffusion constant in water) is the same for most of the molecules tested. There is no extraordinary dependence of Pd on molecular weight; thus, given Pd(acetamide), Pd(1,6 hexanediol) is correctly predicted from the Knc and D values for the two molecules. The major exceptions are H2O, whose value of Pd/DKnc is about 10-fold larger, and urea, whose value is about 5-fold smaller than the general average. In a "tight" membrane such as SC, Pd(n-butyramide)/Pd(isobutyramide)=2.5; thus this bilayer manifests the same sort of discrimination between branched and straight chain molecules as occurs in many plasma membranes. Although the absolute values of the Pd's change by more than a factor of 100 in going from the tightest membrane (SC) to the loosest (L), the relative values remain approximately constant. The general conclusion of this study is that H2O and nonelectrolytes cross lipid bilayer membranes by a solubility-diffusion mechanism, and that the bilayer interior is much more like an oil (a la Overton) than a rubber-like polymer (a la Lieb and Stein).

Outer membrane of Salmonella typhimurium: accessibility of phospholipid head groups to phospholipase c and cyanogen bromide activated dextran in the external medium.

Abstract: Whole cells of Salmonella typhimurium were treated with Bacillus cereus phospholipase C or with CNBr-activated dextran. If phosphatidylethanolamine head groups are exposed and accessible on the outer surface of the outer membrane of these cells, it was expected that these groups would be hydrolyzed by the former agent, and become covalently coupled to the latter agent. With strains producing lipopolysaccharides of S or Rc type, results did not indicate the presence of any accessible head groups on the outer surface. In contrast, with strains that produce outer membranes containing less complete lipopolysaccharides (Rd or Re type) and reduced amounts of proteins, both methods clearly showed the presence of exposed phosphatidylethanolamine head groups. These data can be most easily explained by assuming that the outer membranes of S and Rc strains either contains all phospholipid molecules in its inner leaflet or has proteins that completely cover up the head groups at its outer surface. In either model, the reduction in the amount of outer membrane proteins in Rd or Re mutants would produce membranes with exposed phospholipid head groups. CNBr-activated dextran can be easily prepared, and reacts with high efficiency under near-physiological conditions. Its additional advantage as a nonpenetrating membrane-labeling reagent is that we can be quite confident on its impermeability because of its size, in contrast, with most other reagents whose presumed impermeability is dependent only on the presence of charged groups.

Thermotropic transitions in biomembranes.

Abstract: One of the most prominent characteristics of protein-free lipid bilayers and bio­ membranes alike is their ability to undergo a reversible thermotropic transition from a fluid state at high temperature to a crystalline state at low temperature Such transitions were, in fact, first characterized in phospholipids before they were found in biomembranes They resemble some transitions found in related systems such as soaps in water and share some features in common with the melting of more conventional materials such as hydrocarbons Since the structural framework of bio­ membranes is a lipid bilayer, it is not surprising that a drastic change in its state has important effects upon the life of the cell Hence, although the initial impact of the demonstration of such transitions in membranes (1) was the insight into the structure of the membrane matrix, more recent research has emphasized their physiological implications and has explored their use as a means of understanding lipid-protein associations In this review we, too, begin by discussing transitions in phospholipid model systems, proceed to the physics of transitions in biomembranes, and finally examine their physiological aspects Although cooperative effects have occasionally been suggested to occur between other membrane components such as proteins, only thermotropic lipid transitions of the type described above have been clearly demonstrated to be a general property of membranes Our discussion is limited to such transitions and to those physiological effects upon which they have a direct bearing

Reversible particle movements associated with unstacking and restacking of chloroplast membranes in vitro.

Abstract: Freeze-fracture and freeze-etch techniques have been employed to study the supramolecular structure of isolated spinach chloroplast membranes and to monitor structural changes associated with in vitro unstacking and restacking of these membranes. High-resolution particle size histograms prepared from the four fracture faces of normal chloroplast membranes reveal the presence of four distinct categories of intramembranous particles that are nonrandomly distributed between grana and stroma membranes. The large surface particles show a one to one relationship with the EF-face particles. Since the distribution of these particles between grana and stroma membranes coincides with the distribution of photosystem II (PS II) activity, it is argued that they could be structural equivalents of PS II complexes. An interpretative model depicting the structural relationship between all categories of particles is presented. Experimental unstacking of chloroplast membranes in low-salt medium for at least 45 min leads to a reorganization of the lamellae and to a concomitant intermixing of the different categories of membrane particles by means of translational movements in the plane of the membrane. In vitro restacking of such experimentally unstacked chloroplast membranes can be achieved by adding 2-20 mM MgCl2 or 100-200 mM NaCl to the membrane suspension. Membranes allowed to restack for at least 1 h at room temperature demonstrate a resegregation of the EF-face particles into the newly formed stacked membrane regions to yield a pattern and a size distribution nearly indistinguishable from the normally stacked controls. Restacking occurs in two steps: a rapid adhesion of adjoining stromal membrane surfaces with little particle movement, and a slower diffusion of additional large intramembranous particles into the stacked regions where they become trapped. Chlorophyll a:chlorophyll b ratios of membrane fraction obtained from normal, unstacked, and restacked membranes show that the particle movements are paralleled by movements of pigment molecules. The directed and reversible movements of membrane particles in isolated chloroplasts are compared with those reported for particles of plasma membranes.

Water permeability of isolated cuticular membranes: The effect of cuticular waxes on diffusion of water.

Abstract: The water permeability of astomatous cuticular membranes isolated from Citrus aurantium L. leaves, pear (Pyrus communis L.) leaves and onion (Allium cepa L.) bulb scales was determined before and after extraction of cuticular waxes with lipid solvents. In pear, the permeability coefficients for diffusion of tritiated water across cuticular membranes (CM) prior to extraction [Pd(CM)] decreased by a factor of four during leaf expansion. In all three species investigated Pd(CM) values of cuticular membranes from fully expanded leaves varied between 1 to 2×10-7 cm-3 s-1·Pd(CM) values were not affected by pH. Extraction of cuticular waxes from the membranes increased their water permeability by a factor of 300 to 500. Permeability coefficients for diffusion of THO across the cutin matrix (MX) after extraction [Pd(MX)] increased with increasing pH. Pdvalues were not inversely proportional to the thickness of cuticular membranes. By treating the cutin matrix and cuticular waxes as two resistances acting in series it was shown that the water permeability of cuticles is completely determined by the waxes. The lack of the Pd(CM) values to respond to pH appeared to be due to structural effects of waxes in the cutin matrix. Cuticular membranes from the submerse leaves of the aquatic plant Potamogeton lucens L. were three orders of magnitude more permeable to water than the cuticular membranes of the terrestrial species investigated.

Effect of phloretin on the permeability of thin lipid membranes.

Abstract: Phloretin dramatically increases cation conductances and decreases anion conductances of membranes treated with ion carriers (nonactin, valinomycin, carbonyl-cyanide-m-chlorophenylhydrazone [CCCP], and Hg(C6F5)2) or lipophilic ions (tetraphenylarsonium [tphAs+] and tetraphenylborate [TPhB-]). For example, on phosphatidylethanolamine membranes, 10(-4) M phloretin increases K+ -nonactin and TPhAs+ conductances and decreases CCCP- and TPhB- conductances 10(3)-fold; on lecithin: cholesterol membranes, it increases K+-nonactin conductance 10(5)-fold and decreases CCCP- conductance 10(3)-fold. Similar effects are obtained with p- and m-nitrophenol at 10(-2) M. These effects are produced by the un-ionized form of phloretin and the nitrophenols. We believe that phloretin, which possesses a large dipole moment, adsorbs and orients at the membrane surface to introduce a dipole potential of opposite polarity to the preexisting positive one, thus increasing the partition coefficient of cations into the membrane interior and decreasing the partition coefficient of anions. (Phloretin may also increase the fluidity of cholesterol-containing membranes; this is manifested by its two- to three-fold increase in nonelectrolyte permeability and its asymmetrical effect on cation and anion conductances in cholesterol-containing membranes.) It is possible that pholoretin's inhibition of chloride, urea, and glucose transport in biological membranes results from the effects of these intense intrafacial dipole fields on the translocator(s) of these molecules.

Plasma Membrane Vesiculation: A New Technique for Isolation of Plasma Membranes

Abstract: Monolayer cell cultures of macrophages, monocytes, myoblasts, and density-inhibited and transformed fibroblasts form and release cell surface membrane vesicles following exposure to formaldehyde, related low-molecular-weight aldehydes, and disulfide blocking agents. Vesicles have a unique composition of proteins and lipids. They show enrichment of cholesterol and sphingomyelin content and a seven-to tenfold enrichment of 5'-nucleotidase activity. Vesicles also contain intramembranous particles and show a trilamellar unit membrane and no ultrastructural evidence of contamination with other cytoplasmic organelles. The technique is proposed as a novel method for isolating plasma membrane vesicles from cells in culture.

Phosphate transport into brush-border membrane vesicles isolated from rat small intestine.

Abstract: Uptake of Pi into brush-border membrane vesicles isolated from rat small intestine was investigated by a rapid filtration technique. The following results were obtained. 1. At pH 7.4 in the presence of a NaCl gradient across the membrane (sodium concentration in the medium higher than sodium concentration in the vesicles), phosphate was taken up by a saturable transport system, which was competitively inhibited by arsenate. Phosphate entered the same osmotically reactive space as D-glucose, which indicates that transport into the vesicles rather than binding to the membranes was determined. 2. The amount of phosphate taken up initially was increased about fourfold by lowering the pH from 7.4 to 6.0.3. When Na+ was replaced by K+, Rb+ or Cs+, the initial rate of uptake decreased at pH 7.4 but was not altered at pH 6.0.4. Experiments with different anions (SCN-,Cl-, SO42-) and with ionophores (valinomycin, monactin) showed that at pH 7.4 phosphate transport in the presence of a Na+ gradient is almost independent of the electrical potential across the vesicle membrane, whereas at pH 6.0 phosphate transport involves the transfer of negative charge. It is concluded that intestinal brush-border membranes contain a Na+/phosphate co-transport system, which catalyses under physiological conditions an electroneutral entry of Pi and Na+ into the intestinal epithelial cell. In contrast with the kidney, probably univalent phosphate and one Na+ ion instead of bivalent phosphate and two Na+ ions are transported together.

Changes in membrane currents in bullfrog atrium produced by acetylcholine.

Abstract: 1. A double sucrose-gap voltage-clamp technique has been used to study the effects of acetylcholine on the membrane currents in atrial trabeculae of the bullfrog, Rana catesbeiana. 2. The second, or slow inward (Ca2+/Na+) current, was found to be markedly reduced by concentrations of acetylcholine greater than approximately 2-0 X 10(-8)M. The resulting decrease in net calcium entry provides a straightforward explanation for the negative inotropic action of acetylcholine in atrial muscle. 3. Measurements of membrane resistance near the resting potential showed that relatively high doses of acetylcholine (approximately 10(-7) M) decrease membrane resistance by about twofold. This effect is shown to be the result of an increase in a time-independent background current which appears to be carried mainly by potassium ions. 4. Using appropriate pharmacological techniques, it has been possible to demonstrate: (i) that the peak slow inward current is reduced to about half its initial value before any significant increase in background current occurs; (ii) that even when a sufficient dose of acetylcholine to produce an increase in background current is used, the background current shows inward-going rectification and cannot account for the observed reduction in the slow inward current. 5. No consistent change was observed in the degree of activation of the time-dependent outward membrane currents after application of concentrations of acetylcholine which produced large decreases in the peak slow inward current. 6. These results are discussed in relation to previous electro-physiological and radioisotope studies of the mechanism of the negative inotropic effect of acetylcholine in cardiac muscle.

Lateral transport on cell membranes: mobility of concanavalin A receptors on myoblasts.

Abstract: We report measurements of the lateral mobility of fluorescent labeled concanavalin A receptor complexes on the plasma membrane of cultured myoblasts of rat. Transport rates were measured by observing the recovery of fluorescence in a small region of the cell surface initially photobleached irreversibly by an intense, focused laser light pulse. Under different conditions we measured effective diffusion coefficients of the receptor complexes in the range 8 x 10(-12) less than D less than 3 x 10(-11) cm2/sec which is two orders of magnitude lower than we found for a fluorescent lipid probe, D approximately (8 +/- 3) x 10(-9) cm2/sec. This large difference and the presence of apparently immobile concanavalin A receptors suggests that factors beyond the fluoidity of the phospholipid bilayer membrane matrix control the rate of lateral transport of the complexes. Effective mobilities of the complexes decrease with increases in the valence, dose, and occupation time of the lectin on the membrane. These properties imply an aggregation of the lectin-receptor complexes. Mobilities are not influenced by azide, colchicine or preincubation at low temperature. Cytochalasin B and low temperatures, during the time of measurement, decrease the lateral transport rate.

Involvement of vesicle coat material in casein secretion and surface regeneration.

Abstract: The ultrastructure of the apical zone of lactating rat mammary epithelial cells was studied with emphasis on vesicle coat structures. Typical 40-60 nm ID "coated vesicles" were abundant, frequently associated with the internal filamentous plasma membrane coat or in direct continuity with secretory vesicles (SV) or plasma membrane proper. Bristle coats partially or totally covered membranes of secretory vesicles identified by their casein micelle content. This coat survived SV isolation. Exocytotic fusion of SV membranes and release of the casein micelles was observed. Frequently, regularly arranged bristle coat structures were identified in those regions of the plasma membrane that were involved in exocytotic processes. Both coated and uncoated surfaces of the casein-containing vesicles, as well as typical "coated vesicles", were frequently associated with microtubules and/or microfilaments. We suggest that coat materials of vesicles are related or identical to components of the internal coat of the surface membrane and that new plasma membrane and associated internal coat is produced concomitantly by fusion and integration of bristle coat moieties. Postexocytotic association of secreted casein micelles with the cell surface, mediated by finely filamentous extensions, provided a marker for the integrated vesicle membrane. An arrangement of SV with the inner surface of the plasma membrane is described which is characterized by regularly spaced, heabily stained membrane to membrane cross-bridges (pre-exocytotic attachment plaques). Such membrane-interconnecting elements may represent a form of coat structure important to recognition and interaction of membrane surfaces.