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1.
If a polyhalide concentration gradient exists across a bilayer lipid membrane (BLM), ion pair movement occurs. The term ion pair indicates a lipid soluble complex of cation and anion with stoichiometry dictated by the respective charges. In a mixture of metal halide (MXn, X = I, Cl, Br) and iodine, the ion pair is of the form M(I2X)n. The flux of ion pairs was monitored by measuring the flow of metal ions or polyhalide ions across the BLM. The flux of ion pairs across the BLM depended on cation crystal radius, fluidity of the membrane, strength of the ion pair complex and on the osmotic gradient (i.e., there exists a coupling between water and ion pair fluxes). The relationship between ion pairing and the electrical conductivity of BLM is briefly discussed. 相似文献
2.
Belgrade rats exhibit microcytic, hypochromic anemia and systemic iron deficiency due to a glycine-to-arginine mutation at residue 185 in a metal ion transporter of a divalent metal transporter/divalent cation transporter/solute carrier 11 group A member 2 or 3 (DMT1/DCT1/SLC11A2), a member of the natural-resistance-associated macrophage protein (Nramp) family. By use of rabbit duodenal tissue, a calcein fluorescence assay has previously been developed to assess transport of divalent metal ions across the small-intestinal brush border membrane (BBM). The assay was readily applied here to rat BBM to learn if it detects DMT1 activity. The results demonstrate protein-mediated transport across the BBM of all tested ions: Mn(2+), Fe(2+), and Ni(2+). Transport into BBM vesicles (BBMV) from (b/b) Belgrade rats was below the detection limit. BBMV of +/b origin had substantial activity. The kinetic rate constant for Ni(2+) membrane transport for +/b BBMV was within the range for normal rabbit tissue. Vesicles from +/b basolateral membranes (BLM) showed similar activity to BBMV while b/b BLM vesicles (BLMV) lacked transport activity. Immunoblots using isoform-specific antibodies demonstrated that intestinal levels of b/b DMT1 were increased compared to +/b DMT1, reflecting iron deficiency. Immunoblots on BBMV indicated that lack of activity in b/b vesicles was not due to a failure of DMT1 to localize to the BBMV; an excess of specific isoforms was present compared to +/b BBMV or duodenal extracts. Immunoblots from BLMV also exhibited enrichment in DMT1 isoforms, despite their distinct origin. Immunofluorescent staining of thin sections of b/b and +/b proximal intestines confirmed that DMT1 localized similarly in mutant and control enterocytes and showed that DMT1 isoforms have distinct distributions within intestinal tissue. 相似文献
3.
WE have used bilayer lipid membranes (BLM) as models for biological membranes to study the transport of metal halides as ion-pairs. In the presence of iodine (I2) we find1 that various monovalent and divalent halides can readily move across BLM along a concentration gradient. The rate of transport increases as the size of the cation increases. As the membrane interior is thought to be of a liquid hydrocarbon nature, these results can be related to those of solvent extraction studies2 which showed that various metal halides could be extracted efficiently from aqueous solutions into organic solvents; the extraction efficiency increases as the cation size increases. 相似文献
4.
Membrane lipid asymmetry influences a multitude of cellular functions, including membrane vesiculation, cell division, and lifespan. Most cells retain the bulk of aminophospholipids to the cytosolic membrane leaflet by means of ATP-fuelled flippases or translocases. Converging lines of evidence indicate that members of the P(4) subfamily of P-type ATPases catalyze aminophospholipid transport and create lipid asymmetry in late secretory and endocytic compartments. Yet P-type ATPases usually pump small cations or metal ions. Atomic structures revealed important aspects of the transport mechanism, and sequence homology indicates that this mechanism is conserved throughout the family. Consequently, understanding how P(4) ATPases acquired the ability to translocate phospholipids instead of simple ions has become a major focus of interest. 相似文献
5.
Characterization of sodium transport in Acholeplasma laidlawii B cells and in lipid vesicles containing purified A. laidlawii (Na+-Mg2+)-ATPase by using nuclear magnetic resonance spectroscopy and 22Na tracer techniques.
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S Mahajan R N Lewis R George B D Sykes R N McElhaney 《Journal of bacteriology》1988,170(12):5739-5746
The active transport of sodium ions in live Acholeplasma laidlawii B cells and in lipid vesicles containing the (Na+-Mg2+)-ATPase from the plasma membrane of this microorganism was studied by 23Na nuclear magnetic resonance spectroscopic and 22Na tracer techniques, respectively. In live A. laidlawii B cells, the transport of sodium was an active process in which metabolic energy was harnessed for the extrusion of sodium ions against a concentration gradient. The process was inhibited by low temperatures and by the formation of gel state lipid in the plasma membrane of this organism. In reconstituted proteoliposomes containing the purified (Na+-Mg2+)-ATPase, the hydrolysis of ATP was accompanied by the transport of sodium ions into the lipid vesicles, and the transport process was impaired by reagents known to inhibit ATPase activity. At the normal growth temperature (37 degrees C), this transport process required a maximum of 1 mol of ATP per mol of sodium ion transported. Together, these results provide direct experimental evidence that the (Na+-Mg2+)-ATPase of the Acholeplasma laidlawii B membrane is the cation pump which maintains the low levels of intracellular sodium characteristic of this microorganism. 相似文献
6.
A dynamic 23Na nuclear magnetic resonance (NMR) technique was applied to the exchange system of Na+ ions present inside and outside large unilamellar vesicles at an equivalent concentration. Addition of melittin to phosphatidylcholine vesicles did not induce any detectable Na+ transport across the membrane but subsequent addition of a trace of chlorpromazine or imipramine did induce Na+ transport. Because the formation of a drug-melittin adduct in a solution was detected by 1H NMR, the activation of melittin channels was assumed to originate from the direct interaction of the drug and melittin. 相似文献
7.
Rat liver basolateral plasma membrane (blLPM) vesicles resuspended in 5 mM Mg2(+)-, Ca2(+)-, Mn2(+)- or Co2(+)-containing media exhibited a markedly lower rate of Na(+)-stimulated L-alanine transport. Divalent cation inhibition of L-alanine uptake was dose dependent, and was observed only when the vesicles were pre-loaded with the divalent cations. The presence or absence of the metal ions in the extravesicular incubation media had no effect on L-alanine transport. Conversely, pretreatment of the vesicles with 0.2 mM of either EGTA or EDTA resulted in higher initial rates of L-alanine transport. This stimulation was overcome by addition of excess divalent cation to the vesicle suspension solution. Since these blLPM vesicles are primarily oriented right-side-out, the divalent cation inhibition of L-alanine transport appears to be a result of their interaction with cytosolic components of the cell membrane. Total Na+ flux as measured with 22Na+ was not affected by intravesicular 5 mM Mg2+ or Ca2+, indicating that the inhibition was not due to dissipation of the Na+ gradient. These observations suggest that intracellular divalent cations may serve to modulate L-alanine transport across the liver cell plasma membrane. 相似文献
8.
The effect of membrane dipole potential on gramicidin channel activity in bilayer lipid membranes (BLMs) was studied. Remarkably, it appeared that proton conductance of gramicidin A (gA) channels responded to modulation of the dipole potential oppositely as compared with gA alkali metal cation conductance. In particular, the addition of phloretin, known to reduce the membrane dipole potential, resulted in a decrease in gA proton conductance, on one hand, and an increase in gA alkali metal conductance, on the other hand, whereas 6-ketocholestanol, the agent raising the membrane dipole potential, provoked an increase in gA proton conductance as opposed to a decrease in the alkali metal cation conductance. The peculiarity of the 6-ketocholestanol effect consisted in its dependence on the H(+) concentration. The experiments with the impermeant dipolar compound, phloridzin, showed that the response of proton transport through gramicidin channels to varying the membrane dipole potential did not change qualitatively if the dipole potential of only one monolayer or both monolayers of the BLM was altered. In contrast to gA proton conductance, the single-channel lifetime changed similarly with varying the membrane dipole potential, regardless of the kind of permeant cations (protons or potassium ions). The results of this study could be tentatively accounted for by an assumption that one of the rate-limiting steps of proton conduction through gramicidin channels represents, in fact, movement of negatively charged species (negative ionic defects) across a membrane. 相似文献
9.
Electrically neutral ionophores (nigericin, monencin) incorporated into a planar bilayer lipid membrane (BLM) bring about hydrogen ion gradient formation in the unstirred layers of BLM if a metal ion gradient on the membrane is prepared. Under these conditions a diffusion potential of a hydrogen ion is generated after addition of a protonophore. Cation selectivity of nigericin, monencin and A23187 has been studied by means of electrical potential measurements in the presence of a protonophore and Men+/nH+-exchangers mentioned above. The data on cation selectivity are in a good agreement with the well known results of the direct measurements of metal ion fluxes. This shows that the effect of generation of the potential on BLM in the presence of a protonophore and a Men+/nH+-exchanger can be used for the estimation of electrically neutral ion fluxes through BLM. 相似文献
10.
《Journal of liposome research》2013,23(2):909-936
AbstractPolymer-coated lipid vesicles labeled with either a radionuclide such as technetium-99m or a paramagnetic cation such as gadolinium or manganese, exhibit an extended half-life in the circulation and reduced reticuloendothelial uptake, and are of potential utility as vascular imaging agents for both nuclear medicine and magnetic resonance. For nuclear medicine applications, lipid vesicles may be prepared with radionuclide either attached to the membrane surface by means of a suitable chelate or else encapsulated within the vesicle and offer two principle advantages compared to radiolabeled red blood cells, (i) vesicle can be prepared prior to patient arrival thereby minimizing delays and scheduling difficulties and (ii) known drug interferences are eliminated. The surface-labeling approach is technically more simple and is better suited to the production of vesicles in a pharmaceutically-acceptable form ready for labeling, however encapsulation results in vesicles which exhibit less renal clearance of entrapped label. The limitations of each approach in real clinical practice are not yet evident. For magnetic resonance applications, paramagnetically-labeled vesicles would be a superior vascular marker compared to small molecular weight paramagnetic chelates and may prove useful for blood volume and perfusion measurements. Surface-associated chelates are the approach of choice for a variety of reasons including increased relaxivity and reduced lipid dose compared to vesicles with entrapped paramagnetic chelates. The presence of polymer on the membrane surface has no effect upon die relaxivity of paramagnetic chelates eitiier entrapped widiin the vesicle or bound to the membrane surface. 相似文献
11.
Probes of membrane electrostatics: synthesis and voltage-dependent partitioning of negative hydrophobic ion spin labels in lipid vesicles. 总被引:1,自引:1,他引:0
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Two spin-labeled derivatives of the hydrophobic anion trinitrophenol have been synthesized and characterized in lipid vesicles. In the presence of lipid vesicles, the electron paramagnetic resonance (EPR) spectra of these probes are a composite of both membrane-bound and aqueous populations; as a result, the membrane-aqueous partitioning can be determined from their electron paramagnetic resonance spectra. The effect of transmembrane potentials on the membrane-aqueous partitioning of these spin-labeled hydrophobic ions was examined in phosphatidylcholine vesicles formed by extrusion. Inside positive membrane potentials promote an increase in the binding of these probes that is quantitatively accounted for by a simple thermodynamic model used previously to describe the partitioning of paramagnetic phosphonium ions. The transmembrane migration rates of these ions are dependent on the dipole potential, indicating that these ions transit the membrane in a charged form. The partitioning of the probe is also sensitive to the membrane surface potential, and this dependence is accurately accounted for using the Gouy-Chapman Stern formalism. As a result of the membrane dipole potential, these probes exhibit a stronger binding and a more rapid transmembrane migration rate compared with positive hydrophobic ion spin labels and provide a new set of negatively charged hydrophobic ion probes to investigate membrane electrostatics. 相似文献
12.
Metalloproteins represent a large share of the proteome and many of them contain paramagnetic metal ions. The knowledge, at atomic resolution, of their structure in solution is important to understand processes in which they are involved, such as electron transfer mechanisms, enzymatic reactions, metal homeostasis and metal trafficking, as well as interactions with their partners. Formerly considered as unfeasible, the first structure in solution by nuclear magnetic resonance (NMR) of a paramagnetic protein was obtained in 1994. Methodological and instrumental advancements pursued over the last decade are such that NMR structure of paramagnetic proteins may be now routinely obtained. We focus here on approaches and problems related to the structure determination of paramagnetic proteins in solution through NMR spectroscopy. After a survey of the background theory, we show how the effects produced by the presence of a paramagnetic metal ion on the NMR parameters, which are in many cases deleterious for the detection of NMR spectra, can be overcome and turned into an additional source of structural restraints. We also briefly address features and perspectives given by the use of 13C-detected protonless NMR spectroscopy for proteins in solution. The structural information obtained through the exploitation of a paramagnetic center are discussed for some Cu2+ -binding proteins and for Ca2+ -binding proteins, where the replacement of a diamagnetic metal ion with suitable paramagnetic metal ions suggests novel approaches to the structural characterization of proteins containing diamagnetic and NMR-silent metal ions. 相似文献
13.
Summary Addition of fragmented sarcoplasmic reticulum (SR) vesicles to the aqueous phase of a black lipid membrane (BLM) causes a large increase in BLM conductance within 10 min. The conductance increase is absolutely dependent on three conditions: The presence of at least 0.5mm Ca++, an acidic phospholipid such as phosphatidylserine or diphosphatidylglycerol in the BLM phospholipid mixture, and an osmotic gradient across the SR vesicle membrane, with the internal osmolarity greater than the external. These requirements are identical to conditions under which the fusion of phospholipid vesicles occurs.When the early part of the time course of conductance rise is examined at high sensitivity, the conductance is seen to increase in discrete steps. The probability of a step increases with the concentration of Ca++ in the medium, with the fraction of acidic phospholipid in the BLM, and with the size of the osmotic gradient across the SR vesicle membrane. On the other hand, the average conductance change per step is independent of the above parameters, but varies with the type and concentration of ions present in the aqueous phase. For a given ion, the mean specific conductance per step is independent of the ion's concentration between 10 and 100mm.The probability distribution of the step-conductances agrees well with the distribution of SR vesicle surface areas, both before and after sonication of the vesicles.The evidence indicates that SR vesicles fuse with the BLM, thereby inserting SR membrane conductance pathways into it. Each discrete conductance jump appears to be the result of the fusion of a single SR vesicle with the BLM. This technique may serve as a general method for inserting membrane vesicles into an electrically accessible system. 相似文献
14.
In order to study interactions between ryanodine receptor calcium release (RyR2) channels during excitation-contraction coupling in cardiac muscle, we used bilayer lipid membrane (BLM) and improved the method of cardiac sarcoplasmic vesicle fusion into BLM. We increased fusion gradient for the vesicles, used chloride ions for fusion up to concentration of 1.2 mol/l and fused the vesicles by adding them directly to the forming BLM. Under these conditions, increased probability of fusion of vesicles containing 2-7 ryanodine channels into BLM was observed. Interestingly about 10% of the channels did not gate into BLM independently, but their gating was coupled. At 53 mmol/l calcium solution, two coupled gating channels had double conductance (191 +/- 15 pS) in comparison with the noncoupled channels (93 +/- 10 pS). Activities of the coupled channels were decreased by 5 micromol/l ryanodine and inhibited by 10 micromol/l ruthenium red similarly as single RyR2 channels. We suppose that cardiac sarcoplasmic vesicles contain single as well as coupled RyR2 channels. 相似文献
15.
The effect of phloretin on the carrier-mediated electrically silent ion fluxes through the bilayer lipid membrane (BLM) was studied. The measurements were carried out according to our conventional technique, i.e. electrical potential recording in the presence of a protonophore, and by a new method--direct measurements of pH shifts in the unstirred layers of the BLM by pH microelectrode. Both techniques gave similar results. It was shown that the addition of phloretin increased the rate of cation/H+ exchange induced by nigericin and decreased the rate of anion/OH(-)-exchange induced by tributyltin. The effect of phloretin was higher in the presence of cholesterol in the BLM. Cholesterol decreased the nigericin- and tributyltin-induced fluxes under our experimental conditions. The application of an external voltage to the membrane had no effect on the ion fluxes thereby showing that these fluxes were electroneutral. The most probable explanation of these results bases on the effect of the membrane dipole potential on the electroneutral fluxes of ions. The possible mechanism of the dipole potential effect on the carrier-mediated electrically silent ion fluxes was discussed in terms of two competing hypotheses--the translocation through the membrane or the reactions at the membrane surface being the rate-limiting steps of the whole transport process. 相似文献
16.
17.
The white rot fungus, Phanerochaete chrysosporium, is one of the few organisms with documented ability to degrade lignin. Protoplasts from P. chrysosporium were disrupted by osmotic shock and membrane vesicles were isolated from the cell debris. The vesicles exhibit active glucose transport that is consistent with a glucose/H+ symport mechanism. An artificial gradient of H+ (outside greater than inside) stimulates glucose uptake. Conversely, a glucose gradient (outside greater than inside) results in the accumulation of H+ by the vesicles. Glucose uptake is not stimulated by either a Na+ or a K+ gradient. Furthermore, glucose transport is electrogenic, since glucose uptake may be driven by a membrane potential (negative interior) created by K+ diffusion mediated by valinomycin. 相似文献
18.
There is extensive ultrastructural evidence in endothelium for the presence of chained vesicles or clusters of attached vesicles, and they are considered to be involved in specific transport mechanisms, such as the formation of trans-endothelial channels. However, few details are known about their mechanical characteristics. In this study, the formation mechanism and mechanical aspects of vascular endothelial chained vesicles are investigated theoretically, based on membrane bending strain energy analysis. The shape of the axisymmetric vesicles was computed on the assumption that the cytoplasmic side of the vesicle has a molecular layer or cytoskeleton attached to the lipid bilayer, which induces a spontaneous curvature in the resting state. The bending strain energy is the only elasticity involved, while the shear elasticity is assumed to be negligible. The surface area of the membrane is assumed to be constant due to constant lipid bilayer thickness. Mechanically stable shapes of chained vesicles are revealed, in addition to a cylindrical tube shape. Unfolding of vesicles into a more flattened shape is associated with increase in bending energy without a significant increase in membrane tension. These results provide insights into the formation mechanism and mechanics of the chained vesicle. 相似文献
19.
In the presence of concentration gradient of metal ions on bilayer lipid membrane (BLM) the addition of non-electrogenic carriers results in a formation of concentration gradient of hydrogen ions in the unstirred layers near membrane. Addition of protonophore under these conditions brings about the formation of diffusion potential of hydrogen ions. This effect underlies the method of measuring non-electrogenic fluxes on BLM initiated in the presence of Men+/nH+ - exchangers. The proposed method was tested on the following Men+/nH+ - exchangers: nigericin, monensin and A23187. The order of cationic selectivity of the given carriers obtained by measuring the potentials on BLM in the presence of protonophores agrees with literature data, which were obtained by direct measurements of ionic fluxes. 相似文献
20.
Characterization of an integral protein of the brush border membrane mediating the transport of divalent metal ions 总被引:3,自引:0,他引:3
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The transport of Fe(2+) and other divalent transition metal ions across the intestinal brush border membrane (BBM) was investigated using brush border membrane vesicles (BBMVs) as a model. This transport is an energy-independent, protein-mediated process. The divalent metal ion transporter of the BBM is a spanning protein, very likely a protein channel, that senses the phase transition of the BBM, as indicated by a break in the Arrhenius plot. The transporter has a broad substrate range that includes Mn(2+), Fe(2+), Co(2+), Ni(2+), Cu(2+), and Zn(2+). Under physiological conditions the transport of divalent metal ions is proton-coupled, leading to the acidification of the internal cavity of BBMVs. The divalent metal ion transporter can be solubilized in excess detergent (30 mM diheptanoylphosphatidylcholine or 1% Triton X-100) and reconstituted into an artificial membrane system by detergent removal. The reconstituted membrane system showed metal ion transport characteristics similar to those of the original BBMVs. The properties of the protein described here closely resemble those of the proton-coupled divalent cation transporter (DCT1, Nramp2) described by, Nature. 388:482-488). We may conclude that a protein of the Nramp family is present in the BBM, facilitating the transport of Fe(2+) and other divalent transition metal ions. 相似文献