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1.
Summary Voltage jump-current relaxation experiments have been performed with valinomycin-doped membranes of mixtures of 1,2-dipentadecylmethylidene-glycero-3-phosphorylcholine (PC) and charged-phosphatidic acid (PA). Both relaxation processes predicted by a simple carrier model could be resolved which allowed the calculation of the rate constants of the Rb+ transport. The dependence of the rate constants on the membrane composition indicates that (i) the lipids in the mixed membranes are homogeneously distributed and that (ii) no major difference exists between the composition of the membrane and that of the torus. The analysis of the stationary conductance data, however, shows that the valinomycin content of the mixed membranes depends strongly on their lipid composition. Addition of Ca++ ions to a 11 mixture induces a phase separation into PA domains of very low conductivity and PC-enriched regions of high conductivity. Half saturation is reached atc
ca=5×10–4
m. At 10–2
m Ca++ in the aqueous phase, the rate constants clearly indicate that all PA molecules are electrically passivated and only pure PC domains contribute to the membrane current. A detailed picture is thus derived of the coupling of a model transport system to the externally triggered membrane reorganization. 相似文献
2.
Ca2+-induced phase separation in phosphatidylserine/phosphatidylethanolamine and phosphatidylserine/phosphatidylethanolamine/phosphatidylcholine model membranes was studied using spin-labeled phosphatidylethanolamine and phosphatidylcholine and compared with that in phosphatidylserine/phosphatidylcholine model membranes studied previously. The phosphatidylethanolamine-containing membranes behaved in qualitatively the same way as did phosphatidylserine/phosphatidylcholine model membranes. There were some quantitative differences between them. The degree of phase separation was higher in the phosphatidylethanolamine-containing membranes. For example, the degree of phase separation in phosphatidylserine/phosphatidylethanolamine membranes containing various mole fractions of phosphatidylserine was 94--100% at 23 degrees C and 84--88% at 40 degrees C, while the corresponding value for phosphatidylserine/phosphatidylcholine membranes was 74--85% at 23 degrees C and 61--79% at 40 degrees C. Ca2+ concentration required for the phase separation was lower for phosphatidylserine/phosphatidylethanolamine than that for phosphatidylserine/phosphatidylcholine membranes; concentration to cause a half-maximal phase separation was 1.4 . 10(-7) M for phosphatidylserine-phosphatidylethanolamine and 1.2 . 10(-6) M for phosphatidylserine/phosphatidylcholine membranes. The phase diagram of phosphatidylserine/phosphatidylethanolamine membranes in the presence of Ca2+ was also qualitatively the same as that of phosphatidylserine/phosphatidylcholine except for the different phase transition temperatures of phosphatidylethanolamine (17 degrees C) and phosphatidylcholine (-15 degrees C). These differences were explained in terms of a greater tendency for phosphatidylethanolamine, compared to phosphatidylcholine, to form its own fluid phase separated from the Ca2+-chelated solid-phase phosphatidylserine domain. 相似文献
3.
4.
Single-channel conductance fluctuations are analysed for gramicidin A incorporated into binary-mixed black lipid membranes of charged phosphatidic acid and neutral lecithin in different molar ratios. At very low Ca++ concentrations in the electrolyte (i.e. in the presence of EDTA) homogeneous lipid mixtures are identified through their conductance and life time probability distributions for integral gramicidin pores. As for the pure lipid components, the conductance histograms each show a single maximum with regular width and for all channels a single mean lifetime is found.For Ca++-levels (10-6–10-5
M) that are close to the critical demixing concentration (10-4
M) unusually broad conductance distributions and reduced lifetimes are found provided the PC content, x, of the membrane is close to the critical mixture (x
crit0.5). We interpret this as a first example of the coupling of a membrane function (the transport of ions) to a lipid matrix with locally fluctuating composition close to a critical demixing point.For
the conductance histogram of gramicidin A in an equimolar mixture of PA and PC shows two well-separated maxima. A correlation analysis between conductance and lifetime of the single pores shows that the two channel populations also differ significantly in their mean channel lifetime, *. This finding is interpreted as being direct evidence for Ca++-induced lateral phase separation in black lipid membranes, as has been postulated recently.Abbreviations used HEPES
N-2-hydroxyethyl-piperazine-N-2-ethane-sulfonic acid
- EDTA
ethylenediaminetetraacetic acid 相似文献
5.
6.
The effects of millimeter microwaves in the frequency range of 54–76 GHz on capacitance and conductance of lipid bilayer membranes (BLM) were studied. Some of the membranes were modified by gramicidin A and amphotericin B or by tetraphenylboron anions (TPhB?). The millimeter microwaves were pulse-modulated (PW) at repetition rates ranging from 1 to 100 pps, PW at 1000 pps, or unmodulated continuous waves (CW). The maximum output power at the waveguide outlet was 20 mW. It was found that CW irradiation decreased the unmodified BLM capacitance by 1.2% ± 0.5%. At the same time, membrane current induced by TPhB transport increased by 5% ± 1%. The changes in conductance of ionic channels formed by gramicidin A and amphotericin B were small (0.6% ± 0.4%). No “resonance-like” effects of mm-wave irradiation on membrane capacitance, ionic channel currents, or TPhB transport were detected. All changes in membrane capacitance and currents were independent of the modulation employed and were equivalent to heating by approximately 1.1 °C. © 1995 Wiley-Liss, Inc. 相似文献
7.
M Deleers J P Servais E Wulfert 《Biochemical and biophysical research communications》1986,136(2):476-481
Fluorescence quenching of 1-acyl-2-[6[(7 nitro-2,1,3-benzoxadiazol-4yl) amino]caproyl] phosphatidyl choline in small unilamellar vesicles consisting of phosphatidyl serine has been used to monitor the lipid phase separation induced by Zn2+ and Ca2+. Phase separation of vesicle membranes was observed with Zn2+ at concentrations as low as 125 microM. Low concentrations of Zn2+ required long incubation times to reach maximal quenching (120 minutes at 375 microM). When low concentrations of Ca2+ were added to the preparation during the developing phase of Zn2+-induced quenching, an explosive increase in fluorescence quenching was instantenously observed. Phase separation induced by sub-millimolar concentrations of Ca2+ could be increased at least 4 times when vesicles were pre-incubated with 250 microM of Zn2+. 相似文献
8.
P Caroni P Gazzotti P Vuilleumier W Simon E Carafoli 《Biochimica et biophysica acta》1977,470(3):437-445
The effect of a synthetic neutral ligand on the Ca2+ permeability of several biological membranes has been investigated. The ligand had been previously shown to possess Ca2+ -ionophoric activities in artificial phospholipid membranes. The neutral ionophore is able to transport Ca2+ across the membranes of erythrocytes and sarcoplasmic reticulum, when lipophilic anions such as tetraphenylborate and carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) are present, presumably to facilitate the diffusion of the charged Ca2+ -ionophore complex across the hydrophobic core of the membrane. In mitochondria, the neutral ionophore promotes the active transport of Ca2+ in response to the negative membrane potential generated by respiration, in the presence of the specific inhibitor of the natural carrier ruthenium red. 相似文献
9.
ATP-dependent Ca2+ uptake by subfractions of skeletal muscle sarcoplasmic reticulum (SR) was studied with the Ca2+ indicator dye, antipyrylazo III. Ca2+ uptake by heavy SR showed two phases, a slow uptake phase and a fast uptake phase. By contrast, Ca2+ uptake by light SR exhibited a monophasic time course. In both fractions a steady state of Ca2+ uptake was observed when the concentration of free Ca2+ outside the vesicles was reduced to less than 0.1 microM. In the steady state, the addition of 5 microM Ca2+ to the external medium triggered rapid Ca2+ release from heavy SR but not from light SR, indicating that the heavy fraction contains a Ca2+-induced Ca2+ release channel. During Ca2+ uptake, heavy SR showed a constant Ca2+-dependent ATPase activity (1 mumol/mg protein X min) which was about 150 times higher than the rate of Ca2+ uptake in the slow uptake phase. Ruthenium red, an inhibitor of Ca2+-induced Ca2+ release, enhanced the rate of Ca2+ uptake during the slow phase without affecting Ca2+-dependent ATPase activity. Adenine nucleotides, activators of Ca2+ release, reduced the Ca2+ uptake rate. These results suggest that the rate of Ca2+ accumulation by heavy SR is not proportional to ATPase activity during the slow uptake phase due to the activation of the channel for Ca2+-induced Ca2+ release. In addition, they suggest that the release channel is inactivated during the fast Ca2+ uptake phase. 相似文献
10.
Previous interpretations of the kinetics of transport of hydrophobic ions through membranes have been based on one of three limiting assumptions. Either diffusion in the aqueous phase was taken to be rapid, or ionic motion was constrained to the membrane or a steady state was presumed to be established within the membrane. We present a general treatment of the coupled diffusion process through both the aqueous phase and the membrane; our theory contains the previous results as limiting cases. It is applied to voltage jump-current relaxation experiments on black lipid membranes in the presence of dipicrylamine or sodium tetraphenylborate. We have attempted to establish the rate of desorption from the membrane. For the system phosphatidylserine/tetraphenylborate, the rate of desorption and the rate of translocation were found to be comparable. 相似文献
11.
G V Chernysheva V I Mel'nik G G Amarantova 《Biulleten' eksperimental'no? biologii i meditsiny》1981,92(11):548-549
Thirty-day hypokinesia in Wistar rats did not affect the rate of Ca2+ transport and the activity of Ca-ATPase in light and heavy fractions of sarcoplasmic reticulum of primarily white muscles. As hypokinesia was raised up to 90 days, these indicators increased. The intensity of lipid peroxidation in sarcoplasmic reticulum was lower in hypokinetic animals. 相似文献
12.
The effect of peptide/lipid hydrophobic mismatch on the phase behavior of model membranes mimicking the lipid composition in Escherichia coli membranes
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The effect of hydrophobic peptides on the lipid phase behavior of an aqueous dispersion of dioleoylphosphatidylethanolamine and dioleoylphosphatidylglycerol (7:3 molar ratio) was studied by (31)P NMR spectroscopy. The peptides (WALPn peptides, where n is the total number of amino acid residues) are designed as models for transmembrane parts of integral membrane proteins and consist of a hydrophobic sequence of alternating leucines and alanines, of variable length, that is flanked on both ends by tryptophans. The pure lipid dispersion was shown to undergo a lamellar-to-isotropic phase transition at approximately 60 degrees C. Small-angle x-ray scattering showed that at a lower water content a cubic phase belonging to the space group Pn3m is formed, suggesting also that the isotropic phase in the lipid dispersion represents a cubic liquid crystalline phase. It was found that the WALP peptides very efficiently promote formation of nonlamellar phases in this lipid system. At a peptide-to-lipid (P/L) molar ratio of 1:1000, the shortest peptide used, WALP16, lowered the lamellar-to-isotropic phase transition by approximately 15 degrees C. This effect was less for longer peptides. For all of the WALP peptides used, an increase in peptide concentration led to a further lowering of the phase transition temperature. At the highest P/L ratio (1:25) studied, WALP16 induced a reversed hexagonal liquid crystalline (H(II)) phase, while the longer peptides still promoted the formation of an isotropic phase. Peptides with a hydrophobic length larger than the bilayer thickness were found to be unable to inhibit formation of the isotropic phase. The results are discussed in terms of mismatch between the hydrophobic length of the peptide and the hydrophobic thickness of the lipid bilayer and its consequences for lipid-protein interactions in membranes. 相似文献
13.
We investigate the competing effects of hydrophobic mismatch and chain stretching on the morphology and evolution of domains in lipid membranes via Monte Carlo techniques. We model the membrane as a binary mixture of particles that differ in their preferred lengths, with the shorter particles mimicking unsaturated nonraft lipids and the longer particles mimicking saturated raft lipids. We find that phase separation can be induced upon increasing either the ratio J/kappa of the hydrophobic surface tension J to the compressibility modulus kappa. J/kappa determines the decay length for thickness changes. When this decay length is larger than the system size the membrane remains mixed. Furthermore, increasing the thickness relaxation time can induce transient phase separation. 相似文献
14.
Y Chen 《Biophysical chemistry》1983,17(2):87-95
The effect of transit time on the electrical transport noise of a closed one-barrier model at equilibrium as proposed by Kolb and Läuger [6] is studied using the master-equation approach. A transit time is the time for an ion to cross the energy barrier (membrane interior) when the energy of the ion reaches the barrier height. Both the time correlation function and the noise power spectrum are obtained as functions of the transit time of the ions. Possible effects of transit time on the time correlation function of transport of dipicrylamine ions in lipid bilayers as reported by Bruner and Hall [13] and on the noise power spectrum as reported by Kolb and Läuger [6] are discussed. 相似文献
15.
Sarcoplasmic reticulum vesicles adsorbed on a black lipid membrane generate an electrical current after a fast increment of the concentration of ATP. This demonstrates directly that the sarcoplasmic Ca2+-ATPase from skeletal muscle acts as an electrogenic ion pump. The increment of the concentration of ATP is achieved by the photolysis of caged ATP (P3-1-(2-nitro)phenylethyl adenosine 5'-triphosphate) a protected analogue of ATP (Kaplan, J.H. et al. (1978) Biochemistry 17, 1929-1935), which is split into ATP and 2-nitroso acetophenone. The release of ATP leads to a transient current flow across the lipid membrane indicating that the vesicles are capacitatively coupled to the underlying lipid membrane. In addition to this transient signal, a stationary current flow is obtained in the presence of ionophores which increase the conductance of the bilayer system and prevent the accumulation of Ca2+ in the lumen of the vesicles. The direction of the transient and the stationary current is in accordance with the concept that Ca2+ is pumped into the lumen of the vesicles. The transient current depends on the concentration of ATP, Ca2+ and Mg2+ as would be the case for a current generated by the sarcoplasmic Ca2+-ATPase. Its amplitude is half-maximal at 10 microM ATP and 1 microM Ca2+. At Ca2+ concentrations above 0.1 mM the amplitude of the current signal declines again. The Mg2+ concentration dependence of the current amplitude at a constant ATP concentration indicates that the MgATP complex is the substrate for the activation of the current. The pump current is inhibited by vanadate and ADP. No current signal is observed if caged ATP is replaced by caged ADP. However, the release of ADP from caged ADP generates a pump current in the presence of an ATP generating system such as creatine phosphate and creatine kinase. 相似文献
16.
17.
M Hilge 《The Journal of biological chemistry》2012,287(38):31641-31649
The binding of Ca(2+) to two adjacent Ca(2+)-binding domains, CBD1 and CBD2, regulates ion transport in the Na(+)/Ca(2+) exchanger. As sensors for intracellular Ca(2+), the CBDs form electrostatic switches that induce the conformational changes required to initiate and sustain Na(+)/Ca(2+) exchange. Depending on the presence of a few key residues in the Ca(2+)-binding sites, zero to four Ca(2+) ions can bind with affinities between 0.1 to 20 μm. Importantly, variability in CBD2 as a consequence of alternative splicing modulates not only the number and affinities of the Ca(2+)-binding sites in CBD2 but also the Ca(2+) affinities in CBD1. 相似文献
18.
Fernández-Montalván A Assfalg-Machleidt I Pfeiler D Fritz H Jochum M Machleidt W 《Biological chemistry》2006,387(5):617-627
Mu- and m-calpain are cysteine proteases requiring micro- and millimolar Ca2+ concentrations for their activation in vitro. Among other mechanisms, interaction of calpains with membrane phospholipids has been proposed to facilitate their activation by nanomolar [Ca2+] in living cells. Here the interaction of non-autolysing, C115A active-site mutated heterodimeric human mu-calpain with phospholipid bilayers was studied in vitro using protein-to-lipid fluorescence resonance energy transfer and surface plasmon resonance. Binding to liposomes was Ca2+-dependent, but not selective for specific phospholipid head groups. [Ca2+]0.5 for association with lipid bilayers was not lower than that required for the exposure of hydrophobic surface (detected by TNS fluorescence) or for enzyme activity in the absence of lipids. Deletion of domain V reduced the lipid affinity of the isolated small subunit (600-fold) and of the heterodimer (10- to 15-fold), thus confirming the proposed role of domain V for membrane binding. Unexpectedly, mutations in the acidic loop of the 'C2-like' domain III, a putative Ca2+ and phospholipid-binding site, did not affect lipid affinity. Taken together, these results support the hypothesis that in vitro membrane binding of mu-calpain is due to the exposed hydrophobic surface of the active conformation and does not reduce the Ca2+ requirement for activation. 相似文献
19.
Iu G Rovin 《Biofizika》1978,23(4):638-640
Effect of water-soluble detergents, such as triton X-100, saponin and trimethyl octadecylammonium bromide on the concentration of black spots, tension and stability of black lipid membranes was studied. Changes in mechanic stability of the lipid bilayer are discovered, which are in a good correlation with the litic effect of true detergents on cell membranes. 相似文献
20.
The effect of Ca2+ on the sulfhydryl reactivity of troponin. Evidence for a Ca2+-induced conformational charge 总被引:3,自引:0,他引:3
F Fuchs 《Biochimica et biophysica acta》1971,226(2):453-458