带电磷脂膜泡的内、外表面电荷密度和表面电位

本文按Gouy-Chapman理论,推导了稳定条件下磷脂膜泡膜内、外表面电荷密度/表面电位关系和表面电位分布以及带电磷脂在膜泡内外两侧的不对称分布的近似表达式.并对这些表示式的极限情况和可能的应用进行了讨论,同时对带电磷脂在膜泡膜内、外两侧的不对称分布也作了讨论.     

Structural Features of N-Glycans of Seaweed Glycoproteins: Predominant Occurrence of High-Mannose Type N-Glycans in Marine Plants

An ice nucleation-active strain of Erwinia uredovora shed vesicles when cultured at low temperature (l0?C). We isolated an ice nucleation-active vesicle fraction from the culture medium by ultrafiltration, ultracentrifugation, and gel filtration. Western blot analysis showed that this cell-free vesicle fraction contained an ice nucleation-active protein (InaU). The process of the InaU transport to a shed vesicle was examined by immunohistochemical analysis using electron microscopy. The examination showed the following successive processes: InaU molecules first assemble around the inner membrane, then the assembly enters a vesicle justforming on the surface of the outer membrane, and finally the vesicle, 100–400 nm in diameter, leaves the surface to be shed with InaU molecules occluded.     

The interaction of lanthanide and calcium salts with phospholipid bilayer vesicles: The validity of the nuclear magnetic resonance method for determination of vesicle bilayer phospholipid surface ratios

Contrary to a recent report (B. Sears et al., Biochemistry 15 (1976) 1635), it has been determined that the ratio of the number of phospholipids on the inner and outer surfaces of phospholipid bilayer vesicles can be accurately determined by NMR paramagnetic ion shift reagent studies of vesicles. It is concluded that the metal interacts with all of the phospholipid on the exposed bilayer surface. A ratio of outer phospholipid to inner surface phospholipid of 2.1 ± 0.1 is obtained regardless of the nucleus studies, position of the nucleus relative to the metal ion binding site, molar ratio of metal to phospholipid over three orders of magnitude, or location of the metal ion on the inside or outside of the vesicle. Additionally, P-31 NMR studies using LaCl₃ and CaCl₂ indicate that Ca²⁺ weakly interacts with egg PC vesicles and than the lanthanides are adequate substitutes for Ca²⁺ since neither metal is found to perturb measurably the average polar head group conformation.     

The response of giant phospholipid vesicles to pore-forming peptide melittin

The interaction between the pore-forming peptide melittin (MLT) and giant phospholipid vesicles was explored experimentally. Micromanipulation and direct optical observation of a vesicle (loaded with sucrose solution and suspended in isomolar glucose solution) enabled the monitoring of a single vesicle response to MLT. Time dependences of the vesicle size, shape and the composition of the inner solution were examined at each applied concentration of MLT (in the range from 1 to 60 μg/ml). The response varied with MLT concentration from slight perturbation of the membrane to disintegration of the vesicle. A model for MLT-vesicle interaction is proposed that explains the observed phenomena in the entire span of MLT concentrations and is consistent with deduced underlying mechanisms of MLT action: trans-membrane positioning and dimerization of MLT, the lipid flow from the outer to the inner membrane leaflet induced by MLT translocation, formation of pores and the consequent transport of small molecules through the membrane. The results of the theoretical analysis stress the role of dimers in the MLT-membrane interaction and demonstrate that the MLT-induced membrane permeability for sugar molecules in this experimental set-up depends on both MLT concentration and time.     

The possible role of fixed membrane surface charges in acetylcholine release at the frog neuromuscular junction

Summary Bass and Moore [Proc. Nat. Acad. Sci. 55:1214 (1966)] proposed that the vesicles containing acetylcholine undergo Brownian motion in the nerve terminals. Acetylcholine is released whenever a vesicle touches the inner face of the axolemma of the nerve terminal. The frequency at which contact is made is limited by an energy barrier that must be overcome before the vesicle can touch the axolemma. The energy barrier has two components. (1) An electrostatic repulsion between positive, fixed charges on the vesicles and a relatively positive potential at the face of the axolemma that is generated by the resting potential. (2) A layer of water molecules held to the vesicle by the surface charge. This model is inconsistent with experimental data. A modification of the model is presented. Both the vesicle and the inner face of the axolemma are assumed to have fixed, negative surface charges that are responsible for the energy barrier. By a series of simplifications, the model leads to two predictions. (1) A plot of the ln (miniature end plate potentials/sec) as a function of the concentration of ions in the axoplasm)^–0.5 should give a straight line. (2) A plot of ln (end plate potential amplitudes) as a function of (extracellular Ca⁺⁺ concentration)^–0.5 should give a straight line. These predictions are shown to agree reasonably well with experimental data.     

Surface distribution of the fatty acid side chains of phosphatidylethanolamine in mixed phospholipid vesicles

A method has been developed for the selective determination of the fatty acid side chain distribution associated with the amino containing phospholipids located in the inner and outer surfaces of membranes. Using sonicated phosphatidylethanolamine/phosphatidylcholine vesicles as a model, the analysis consists of selective labeling of the outer surface amino groups with the membrane impermeable reagent 2,4,6-trinitrobenzenesulfonic acid. Outer and inner surface phosphatidylethanolamine fractions are separated by thin-layer chromatography. Analysis of methyl esters derived from these two fractions, by gas-liquid chromatography, yields the fatty acid side chain distribution. Our results show that there is no mol fraction dependence of the incorporation of any specific fatty acid side chains of egg yolk phosphatidylethanolamine into the vesicle or any preferential distribution of these side chains in the inner or outer vesicle surface. The surface distribution of the egg yolk phosphatidylethanolamine molecules in these vesicles appears to be determined by the head group packing requirements and not the fatty acid side chain composition.     

Surface distribution of the fatty acid side chains of phosphatidylethanolamine in mixed phospholipid vesicles.

A method has been developed for the selective determination of the fatty acid side chain distribution associated with the amino containing phospholipids located in the inner and outer surfaces of membranes. Using sonicated phosphatidylethanolamine/phosphatidylcholine vesicles as a model, the analysis consists of selective labeling of the outer surface amino groups with the membrane impermeable reagent 2,4,6-trinitrobenzenesulfonic acid. Outer and inner surface phosphatidylethanolamine fractions are separated by thin-layer chromatography. Analysis of methyl esters derived from these two fractions, by gas-liquid chromatography, yields the fatty acid side chain distribution. Our results show that there is no mol fraction dependence of the incorporation of any specific fatty acid side chains of egg yolk phosphatidylethanolamine into the vesicle or any preferential distribution of these side chains in the inner or outer vesicle surface. The surface distribution of the egg yolk phosphatidylethanolamine molecules in these vesicles appears to be determined by the head group packing requirements and not the fatty acid side chain composition.     

Topology of amino-phospholipids in the red cell membrane

The red cell membrane has an asymmetric arrangement of phospholipids. The amino-phospholipids are localized primarily on the inner surface of the membrane and the choline phospholipids are localized to a large extent on the outer surface of the membrane. Evidence is presented based on the use of covalent chemical probes in sequence that the red cell membrane contains heterogeneous domains of PE and PS and that the transport systems for Pi and K⁺ are asymmetrically arranged. Certain amino groups of PE, PS, and/or protein localized on the outer membrane surface are involved in Pi transport and certain amino groups of PE, PS, and/or protein localized on the inner surface of the membrane are involved in K⁺ transport. Cross-linking studies with DFDNB show that the cross-linked PE-PE molecules are rich in plasmalogens. This suggests that clusters of plasmalogen forms of PE occur in the membrane. Both PE and PS are cross-linked to membrane protein. These PE and PS molecules contain 24–28% 16:0 and 18:0 fatty acids and 12% fatty aldehydes. PE and PS molecules are cross-linked to a spectrin-rich fraction. It is proposed that the binding of spectrin to membrane PE and PS may help anchor spectrin to the inner surface of the membrane and regulate shape changes in the cell. K⁺-valinomycin forms a complex with TNBS and converts it from a non-penetrating proble to a penetrating probe. Valinomycin enhances K⁺ leak and Pi leak in the red cells. SITS inhibits completely the valinomycin-induced Pi leak and inhibits partially the valinomycin induced K⁺ leak. Valinomycin and IAA have additive effects on Pi leak. Ouabin has no effect on basal or valino-mycin-induced Pi leak. These data suggest that Pi leak and K⁺ leak occur by separate transport systems. In summary, the amino-phospholipids in the red cell membrane are asymmetrically arranged; some occur in clusters and some are closely associated with membrane proteins. Amino-phospholipids also are believed to bind spectrin to the inner surface of the membrane and also may play a role in cation and anion leak.     

The response of giant phospholipid vesicles to pore-forming peptide melittin

The interaction between the pore-forming peptide melittin (MLT) and giant phospholipid vesicles was explored experimentally. Micromanipulation and direct optical observation of a vesicle (loaded with sucrose solution and suspended in isomolar glucose solution) enabled the monitoring of a single vesicle response to MLT. Time dependences of the vesicle size, shape and the composition of the inner solution were examined at each applied concentration of MLT (in the range from 1 to 60 microg/ml). The response varied with MLT concentration from slight perturbation of the membrane to disintegration of the vesicle. A model for MLT-vesicle interaction is proposed that explains the observed phenomena in the entire span of MLT concentrations and is consistent with deduced underlying mechanisms of MLT action: trans-membrane positioning and dimerization of MLT, the lipid flow from the outer to the inner membrane leaflet induced by MLT translocation, formation of pores and the consequent transport of small molecules through the membrane. The results of the theoretical analysis stress the role of dimers in the MLT-membrane interaction and demonstrate that the MLT-induced membrane permeability for sugar molecules in this experimental set-up depends on both MLT concentration and time.     

The relationship of protein and lipid synthesis during the biogenesis of mitochondrial membranes

Summary Rat liver mitochondria were fractionated into inner and outer membrane components at various times after the intravenous injection of¹⁴C-leucine or¹⁴C-glycerol. The time curves of protein and lecithin labeling were similar in the intact mitochondria, the outer membrane fraction, and the inner membrane fraction. In rat liver slices also, the kinetics of³H-phenylalanine incorporation into mitochondrial KCl-insoluble proteins was identical to that of¹⁴C-glycerol incorporation into mitochondrial lecithin. These results suggest a simultaneous assembly of protein and lecithin during membrane biogenesisThe proteins and lecithin of the outer membrane were maximally labeledin vivo within 5 min after injection of the radioactive precursors, whereas the insoluble proteins and lecithin of the inner membrane reached a maximum specific acitivity 10 min after injection.Phospholipid incorporation into mitochondria of rat liver slices was not affected when protein synthesis was blocked by cycloheximide, puromycin, or actinomycin D. The injection of cycloheximide 3 to 30 min prior to¹⁴C-choline did not affect thein vivo incorporation of lecithin into the mitochondrial inner or outer membranes; however treatment with the drug for 60 min prior to¹⁴C-choline resulted in a decrease in lecithin labeling. These results suggest that phospholipid incorporation into membranes may be regulated by the amount of newly synthesized protein available.When mitochondria and microsomes containing labeled phospholipids were incubated with the opposite unlabeled fractionin vitro, a rapid exchange of phospholipid between the microsomes and the outer membrane occurred. A slight exchange with the inner membrane was observed.     

Carbon-13 nuclear magnetic resonance studies of cerebroside derivatives and their properties in lecithin bilayers (1)

The ¹³C NMR chemical shifts and spin-lattice relaxation times of D-galactosylsphingosine derivatives in CDCl₃-CD₃OD and in egg-yolk lecithin vesicles in D₂O, and of N-acetylpsychosine micelles, are reported. Results with sonicated, unilamellar vesicles containing cerebroside and EYL^a show that (1) cerebrosides decrease the fluidity of the lecithin bilayer membrane and have the greatest effect on the glycerol backbone and choline methyl carbons. (2) N-acetylpsychosine experiences a greater freedom of motion in the galactose region than does cerebroside and does not reduce the fluidity of the lecithin as much as cerebroside. (3) Ac-Psy/EYL vesicles formed are permeable to Yb³⁺ but cerebroside/lecithin vesicles are not. (4) The choline groups on the inner bilayer surface are less mobile than those on the outer surface according to preliminary T₁ measurements of the Yb³⁺-separated resonances. (5) Yb³⁺-induced chemical shifts of choline methyl and choline $\text{C}\text{H}{}_2$OP peaks in mixed cerebroside-lecithin vesicle systems indicate a small preference for cerebroside in the outside monolayer. The data show that these molecules have significant effects on bilayer conformational mobilities, particularly near the surface, and thus demonstrate one mechanism for modulation of cell surface properties by glycosphingolipids.     

THE EXPERIMENTAL PRODUCTION OF DOUBLE PEAKS IN CHARA ACTION CURVES AND THEIR RELATION TO THE MOVEMENT OF POTASSIUM

The action curve in Chara seems to depend (as in Nitella) on the outward movement of K⁺ from the sap. Presumably the increase in permeability in the inner protoplasmic surface and the outward movement of K⁺ destroy the concentration gradient of K⁺ across the inner protoplasmic surface. Hence the outwardly directed P.D. disappears, causing the rise (spike) of the action curve. The outer protoplasmic surface is normally insensitive to K⁺. But when it is made sensitive to K⁺ by treatment with guanidine the outwardly moving K⁺ sets up a positive P.D. on reaching the outer surface and this causes the action curve to fall, producing a peak. Then the curve has 2 peaks, the second being due to the process of recovery. The action curve thus comes to resemble that of Nitella in which the outer protoplasmic surface is normally sensitive to K⁺.     

The Role of Membrane Lateral Tension in Calcium-Induced Membrane Fusion

Calcium-induced fusion of liposomes was studied with a view to understand the role of membrane tension in this process. Lipid mixing due to fusion was monitored by following fluorescence of rhodamine-phosphatidyl-ethanolamine incorporated into liposomal membrane at a self-quenching concentration. The extent of lipid mixing was found to depend on the rate of calcium addition: at slow rates it was significantly lower than when calcium was injected instantly. The vesicle inner volume was then made accessible to external calcium by adding calcium ionophore A23187. No effect on fusion was observed at high rates of calcium addition while at slow rates lipid mixing was eliminated. Fusion of labeled vesicles with a planar phospholipid membrane (BLM) was studied using fluorescence microscopy. Above a threshold concentration specific for each ion, Ca²⁺, Mg²⁺, Cd²⁺ and La³⁺ induce fusion of both charged and neutral membranes. The threshold calcium concentration required for fusion was found to be dependent on the vesicle charge, but not on the BLM charge. Pretreatment of vesicles with ionophore and calcium inhibited vesicle fusion with BLM. This effect was reversible: chelation of calcium prior to the application of vesicle to BLM completely restored their ability to fuse. These results support the hypothesis that tension in the outer monolayer of lipid vesicle is a primary reason for membrane destabilization promoting membrane fusion. How this may be a common mechanism for both purely lipidic and protein-mediated membrane fusion is discussed. Received: 27 September 1999/Revised: 22 March 2000     

Temperature-dependent stomatal movement in tulip petals controls water transpiration during flower opening and closing

Temperature‐dependent tulip petal opening and closing movement was previously suggested to be regulated by reversible phosphorylation of a plasma membrane aquaporin ( Azad et al., 2004a ). Stomatal apertures of petals were investigated during petal opening at 20°C and closing at 5°C. In completely open petals, the proportion of open stomata in outer and inner surfaces of the same petal was 27 ± 6% and 65 ± 3%, respectively. During the course of petal closing, stomatal apertures in both surfaces reversed, and in completely closed petals, the proportion of open stomata in outer and inner surfaces of the same petal was 74 ± 3% and 29 ± 6%, respectively, indicating an inverse relationship between stomatal aperture in outer and inner surfaces of the petal during petal opening and closing. Both petal opening and stomatal closure in the outer surface of the petal was inhibited by a Ca²⁺ channel blocker and a Ca²⁺ chelator, whereas the inner surface stomata remained unaffected. On the other hand, sodium nitroprusside, a nitric oxide donor, had no effect on stomatal aperture of the outer surface but influenced the inner surface stomatal aperture during petal opening and closing, suggesting different signalling pathways for regulation of temperature‐dependent stomatal changes in the two surfaces of tulip petals. Stomata were found to be differentially distributed in the bottom, middle and upper parts of tulip petals. During petal closing, water transpiration was observed by measuring the loss of ³H₂O. Transpiration of ³H₂O by petals was fivefold greater in the first 10 min than that found after 30 min, and the transpiration rate was shown to be associated with stomatal distribution and aperture. Thus, the stomata of outer and inner surfaces of the petal are involved in the accumulation and transpiration of water during petal opening.     

Mechanisms of transmembrane cation transport studied by nuclear magnetic resonance spectroscopy

Several molecules like ionophores, vitamins, ion-binding cyclic peptides, acidic phospholipids, surfactants are known to expose the inner side of vesicles, to the externally added cations. Whereas ionophores and certain other systems bring about these changes by a selective transport (influx) of the cation by specialized mechanisms known as the carrier and channel mechanism, other systems cause lysis and vesicle fusion. These systems have been successfully studied using¹H,³¹ P and¹³C nuclear magnetic resonance spectroscopy after the demonstration, fifteen years ago, of the ability of paramagnetic lanthanide ions to distinguish the inside of the vesicle from the outside. The results of these ’nuclear magnetic resonance kinetics’ experiments are reviewed.     

Exchange and interactions between lipid layers at the surface of a liposome solution.

Lipid organization and lipid transport processes occurring at the air-water interface of a liposome (lipid vesicle) solution are studied by conventional surface pressure-area measurements and interpreted by an adequate theory. At the interface of a dioleoyl phosphatidylcholine vesicle solution, used for demonstration, a well defined two layer structure selfassembles: vesicles disintegrate at the interface forming a surface-adsorbed lipid monolayer, which prevents further disintegration beyond about 1 dyne/cm surface pressure. A layer of vesicles now assembles in close association with the monolayer. This layer is in vesicle diffusion exchange with the solution and in lipid exchange with the monolayer. The lipid exchange occurs exclusively between the monolayer and the outer lipid layer of the vesicles; it is absent between outer and inner vesicle layers. Equilibration of the lipid density in the monolayer with that in the vesicle outer layer provides a coherent and quantitative explanation of the observed hysteresis effects and equilibrium states. The correspondence between monolayer and vesicle outer layer is traced down to equilibrium constants and rate constants and their dependences on surface pressure, vesicle size and concentration. Other alternate realizations of surface structure and exchange, including induced lipid flip-flop within vesicles or vesicle monolayer adhesion or fusion are potential applications of the proposed analysis.     

Exchange and interactions between lipid layers at the surface of a liposome solution

Lipid organization and lipid transport processes occurring at the air-water interface of a liposome (lipid vesicle) solution are studied by conventional surface pressure-area measurements and interpreted by an adequate theory. At the interface of a dioleoyl phosphatidylcholine vesicle solution, used for demonstration, a well defined two layer structure selfassembles: vesicles disintegrate at the interface forming a surface-adsorbed lipid monolayer, which prevents further disintegration beyond about 1 dyne/cm surface pressure. A layer of vesicles now assembles in close association with the monolayer. This layer is in vesicle diffusion exchange with the solution and in lipid exchange with the monolayer. The lipid exchange occurs exclusively between the monolayer and the outer lipid layer of the vesicles; it is absent between outer and inner vesicle layers. Equilibration of the lipid density in the monolayer with that in the vesicle outer layer provides a coherent and quantitative explanation of the observed hysteresis effects and equilibrium states. The correspondence between monolayer and vesicle outer layer is traced down to equilibrium constants and rate constants and their dependences on surface pressure, vesicle size and concentration. p] Other alternate realizations of surface structure and exchange, including induced lipid flip-flop within vesicles or vesicle monolayer adhesion or fusion are potential applications of the proposed analysis.     

FINE STRUCTURE OF THE SPERMATOZOON OF HYDROIDES HEXAGONUS (ANNELIDA), WITH SPECIAL REFERENCE TO THE ACROSOMAL REGION

This paper describes in some detail the structure of the acrosomal region of the spermatozoon of Hydroides as a basis for subsequent papers which will deal with the structural changes which this region undergoes during fertilization. The material was osmium-fixed and mild centrifugation was used to aggregate the spermatozoa from collection to final embedding. The studies concern also the acrosomal regions of frozen-thawed sperm prepared by a method which previously had yielded extracts with egg membrane lytic activity. The plasma membrane closely envelops four readily recognizable regions of the spermatozoon: acrosomal, nuclear, mitochondrial, and flagellar. The acrosome consists of an acrosomal vesicle which is bounded by a single continuous membrane, and its periphery is distinguishable into inner, intermediate, and outer zones. The inner and intermediate zones form a pocket into which the narrowed apex of the nucleus intrudes. Granular material adjoins the inner surface of the acrosomal membrane, and this material is characteristically different for each zone. Centrally, the acrosomal vesicle is spanned by an acrosomal granule: its base is at the inner zone and its apex at the outer zone. The apex of the acrosomal granule flares out and touches the acrosomal membrane over a limited area. In this limited area the adjoining granular material of the outer zone is lacking. The acrosomal membrane of the inner zone is invaginated into about fifteen short tubules. The acrosomal membrane of the outer zone is closely surrounded by the plasma membrane. At the apex of the acrosomal region a small apical vesicle is sandwiched between the plasma membrane and the acrosomal membrane. Numerous frozen-thawed specimens and occasional specimens not so treated show acrosomal regions at the apex of which there is a well defined opening or orifice. Around the rim or lip of this orifice plasma and acrosomal membranes may even be fused into a continuum. The evidence indicates that the apical vesicle and the parts of the plasma and acrosomal membranes which surround it constitute a lid, and the rim of this lid constitutes a natural "fracture line" or rim of dehiscence. Should fracture occur, the lid would be removed and the acrosomal vesicle would be open to the exterior.     

Microelectrode studies of the effect of lanthanum on the electrical potential and resistance of outer and inner cell membranes of isolated frog skin

Summary Microelectrodes were used to investigate the effect of 0.5mm mucosal lanthanum (La³⁺) on the intracellular potential and the resistance of outer and inner isolated frog skin (Rana esculenta) cell membranes. Under short-circuit conditions, the transapical membrane potentialV _o ^sc (mean value=–65.4±3.2 mV, inside negative) hyperpolarized to –108.7±2.3 mV in control skins, after addition of the sodium blocker amiloride. Current-voltage curves for the outer and inner membranes were constructed from the amiloride-inhibitable current versus the outer membrane potentialV _o or the inner membrane potentialV _t . The outer, and to a lesser degree the inner, membrane showed a characteristic nonlinearity with two slope resistances. Addition of La³⁺ to the outer medium increased the short-circuit current to 190% of the control value.V _o ^sc concomitantly changed to –28±3.5 mV and outer and inner membrane resistances fell, considerably attenuating the nonlinearity seen in control skins. La³⁺ is suggested to raise the conductance by its effect on the surface potential. A secondary long-term inhibitory effect of La³⁺ on short-circuit current has been observed. It is ascribed to the penetration of La³⁺ into the sodium channels.     

DIFFERING RATES OF DEATH AT INNER AND OUTER SURFACES OF THE PROTOPLASM : I. EFFECTS OF FORMALDEHYDE ON NITELLA

When protoplasm dies it becomes completely and irreversibly permeable and this may be used as a criterion of death. On this basis we may say that when 0.2 M formaldehyde plus 0.001 M NaCl is applied to Nitella death arrives sooner at the inner protoplasmic surface than at the outer. If, however, we apply 0.17 M formaldehyde plus 0.01 M KCl death arrives sooner at the outer protoplasmic surface. The difference appears to be due largely to the conditions at the two surfaces. With 0.2 M formaldehyde plus 0.001 M NaCl the inner surface is subject to a greater electrical pressure than the outer and is in contact with a higher concentration of KCl. In the other case these conditions are more nearly equal so that the layer first reached by the reagent is the first to become permeable. The outer protoplasmic surface has the ability to distinguish electrically between K⁺ and Na⁺ (potassium effect). Under the influence of formaldehyde this ability is lost. This is chiefly due to a falling off in the partition coefficient of KCl in the outer protoplasmic surface. At about the same time the inner protoplasmic surface becomes completely permeable. But the outer protoplasmic surface retains its ability to distinguish electrically between different concentrations of the same salt, showing that it has not become completely permeable. After the potential has disappeared the turgidity (hydrostatic pressure inside the cell) persists for some time, probably because the outer protoplasmic surface has not become completely permeable.