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1.
1. The influence of Ca2+ on the polymorphic phase behaviour of cardiolipin has been investigated employing 31P NMR and freeze-fracture techniques. The close correlation between the results obtained here and previous X-ray studies (Rand, R.P. and Sengupta, S. (1972) Biochim. Biophys. Acta 255, 484--492) confirms 31P NMR as a useful analytical procedure for investigating the polymorphic phase behaviour of hydrated phospholipids. 2. Ca2+ induces formation of the hexagonal (H11) phase via an intermediary phase which is observed at Ca2+/cardiolipin ratios of less than 1 (mol/mol). This intermediary appears to consist of "inverted' structure which lies adjacent to regions of bilayer structure. 3. The local anaesthetics dibucaine and chlorpromazine produce similar phase changes for cardiolipin as does Ca2+. It is suggested that the anaesthetics interact with the membrane in their charged form and induce their effects by charge neutralization. 相似文献
2.
Syt1 (synaptotagmin 1) is a major Ca2+ sensor for synaptic vesicle fusion. Although Syt1 is known to bind to SNARE (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor) complexes and to the membrane, the mechanism by which Syt1 regulates vesicle fusion is controversial. In the present study we used in vitro lipid-mixing assays to investigate the Ca2+-dependent Syt1 function in proteoliposome fusion. To study the role of acidic lipids, the concentration of negatively charged DOPS (1,2-dioleoyl-sn-glycero-3-phospho-L-serine) in the vesicle was varied. Syt1 stimulated lipid mixing by 3-10-fold without Ca2+. However, with Ca2+ there was an additional 2-5-fold enhancement. This Ca2+-dependent stimulation was observed only when there was excess PS (phosphatidylserine) on the t-SNARE (target SNARE) side. If there was equal or more PS on the v-SNARE (vesicule SNARE) side the Ca2+-dependent stimulation was not observed. We found that Ca2+ at a concentration between 10 and 50?μM was sufficient to give rise to the maximal enhancement. The single-vesicle-fusion assay indicates that the Ca2+-dependent enhancement was mainly on docking, whereas its effect on lipid mixing was small. Thus for Syt1 to function as a Ca2+ sensor, a charge asymmetry appears to be important and this may play a role in steering Syt1 to productively trans bind to the plasma membrane. 相似文献
3.
The effect of the local anesthetics SKF 525-A, dibucaine, tetracaine, procaine, and benzocaine on sarcoplasmic reticulum vesicles was studied. All the anesthetics tested inhibited the phosphorylation of the Ca(2+)-ATPase by Pi in a competitive manner. Tertiary amine and positively charged anesthetics, in addition to competing with Pi, also decreased the apparent affinity of the ATPase for Mg2+. There was a good correlation between the octanol/water partition coefficients and the inhibitory activity of the different anesthetics. All the anesthetics tested induced a 5- to 10-fold increase in the rate of Ca2+ efflux. This was promoted by the same drug concentration that inhibited the phosphorylation of the ATPase by Pi. The effect on Ca2+ efflux was antagonized by the ligands of the ATPase (Mg2+, K+, Ca2+, MgATP, and ADP) and by the organic polyamines ruthenium red, spermine, spermidine, and putrescine. The natural anion heparin was found to potentiate the effect of the positively charged anesthetics on the rate of Ca2+ efflux. It is concluded that the local anesthetics increase the Ca2+ efflux through a nonenergized state of the Ca(2+)-ATPase, rather than promoting a nonspecific Ca2+ leakage through the membrane. 相似文献
4.
M Deleers J P Servais E Wulfert 《Biochemical and biophysical research communications》1986,137(1):101-107
Resonance Energy Transfer between N-(7-nitro-2,1,3 benzoxadiazol -4 yl) phosphatidyl ethanolamine and N-Lissamine-Rhodamine B sulfonyl) phosphatidyl ethanolamine embedded in two different populations of small unilamellar vesicles made of phosphatidyl serine has been used to study the fusion process induced by Zn2+ and Ca2+. Lipid intermixing demonstrating fusion of liposome membranes can already be observed at 125 and 250 mumol/l of Zn2+. After short time pre-incubations with micromolar concentrations of Zn2+ as low as 150 mumol/l, Ca2+ induces an instantaneous increase of vesicle fusion. The lipid intermixing induced by micromolar concentrations of Ca2+ (250-500 mumol/l) could be increased up to 4 times when pre-incubated with 150 or 200 mumol/l of Zn2+. The effect of 1 mM of Ca2+ alone on lipid intermixing can be mimicked by 150 mumol/l of Zn2+ followed by 500 mumol/l of Ca2+. Our data demonstrate that Zn2+ and Ca2+ act synergistically to affect cation-induced membrane fusion. We suggest that Zn2+ specifically alters the physical state of phospholipid membranes making them more prone to calcium-triggered fusion. 相似文献
5.
1. A number of local anaesthetics was shown to inhibit rat liver cholesterol esterase activity towards radioactively labelled cholesterol oleate. The anaesthetics inhibited in the order dibucaine greater than chlorpromazine greater than tetracaine greater than benzocaine greater than procaine greater than lidocaine greater than cocaine. 2. The mode of inhibition was seen to be non-competitive with respect to the substrate and is probably independent of any involvement of Ca2+. 3. The inhibition by tetracaine is partially reversed by sodium deoxycholate. However, all ionic and non-ionic detergents studied, sodium deoxycholate, sodium taurocholate, Triton X-100, and cetyltrimethylammonium bromide are capable of inhibiting the rat liver cholesterol esterase in a concentration dependent manner. Only sodium taurocholate stimulates enzymic activity. 相似文献
6.
ATP-dependent interaction of propranolol and local anaesthetic with sarcoplasmic reticulum. Stimulation of Ca2+ efflux.
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V Shoshan-Barmatz 《The Biochemical journal》1988,256(3):733-739
Preincubation of sarcoplasmic reticulum (SR) with propranolol or tetracaine inhibits Ca2+ accumulation and stimulates ATPase activity by more than 2-fold. This effect is obtained only when the preincubation is carried out in the presence of ATP or other nucleoside triphosphates. The (ATP + drug)-induced inhibition of Ca2+ accumulation is pH-dependent, increasing as the pH rises above 7.5. The presence of micromolar concentrations of Ca2+ or Mg2+ during the preincubation prevents the inhibitory effect of ATP plus drug on Ca2+ accumulation or ATPase activity. The (ATP + drug) modification of SR vesicles resulted in stimulation of a rapid Ca2+ efflux from passively loaded vesicles. The ATP-dependent inhibition of Ca2+ accumulation by the drug is obtained with other local anaesthetics. The drug concentration required for 50% inhibition was 0.15 mM for dibucaine and 0.4 mM for both propranolol and tetracaine, whereas it was 5 mM, 8 mM and greater than 10 mM for lidocaine, benzocaine and procaine respectively. The heavy SR vesicles were only slightly affected by the incubation with propranolol or tetracaine in the presence of ATP, but their sensitivity increased markedly after storage at 0 degrees C for 24-48 h. These results suggest that propranolol and some local anaesthetics, in the presence of ATP, stimulate Ca2+ efflux by modifying a protein factor(s) rather than the phospholipid bilayer. 相似文献
7.
M A Babizhayev 《Archives of biochemistry and biophysics》1988,266(2):446-451
Mechanisms underlying Ca2+ effects on lipid peroxidation (LPO) induced in liposomes (from egg yolk lecithin) and ufasomes (from linolenic acid and methyl linolenate) with the aid of an O2-(.) -generating system (Fe2+ + ascorbate) were studied. It was shown that stimulation of LPO by low Ca2+ concentrations (10(-6)-10(-5)M) was due to its ability to release Fe2+ ions bound to negatively charged (phosphate or carboxylic) lipid groups (of lecithin or linolenic acid), thus increasing the concentration of catalytically active Fe2+. The inhibitory effect of high Ca2+ concentrations was caused by its interaction with superoxide anion radicals and was not observed in LPO systems independent of O2- generation (e.g., Fe2+ + cumol hydroperoxide). 相似文献
8.
V M Savov M A Babizhaev V E Kagan 《Biulleten' eksperimental'no? biologii i meditsiny》1986,101(6):693-695
Mechanisms underlying Ca2+ effects on lipid peroxidation (LPO) induced in liposomes (from egg yolk lecithin) and UFsomes (from linolenic acid, methyl linolenate) with the aid of O2- -system (Fe2+ + ascorbate) were studied. It was shown that stimulation of lipid peroxidation by low Ca2+ concentrations (10(-6)-10(-5) M) was due to its ability to release Fe2+-ions bound to negatively charged (phosphate, carboxylic) lipid groups (of licethin, linolenic acid), thus increasing the concentration of catalytically active Fe2+. The inhibitory effect of high Ca2+ concentrations was caused by its interaction with superoxide anion-radicals and was not observed in LPO-systems, independent of O2- generation (e. g. Fe2+ + cumol hydroperoxide). 相似文献
9.
The effects of local anaesthetics lidocaine, benzocaine, carbisocaine and carbisocaine derivatives, KaQ-7 and Ka-O, in perturbing bovine brain lipid membranes or egg lecithin membranes were compared at pH 6.0; 7.0; and 8.0. The electron spin resonance method with stearic acid labeled at carbon position 16 as the spin probe was employed. The perturbation effects of lidocaine and Ka-O were found to increase with increasing pH of the sample, whereas the effect of carbisocaine decreased with increasing pH. The perturbation effects of benzocaine and KaQ-7 were independent of pH. The pH-dependent perturbation effects of the local anaesthetics tested on lipid membrane fairly corresponded with their pH-dependent potency to block nerve action potentials. 相似文献
10.
Wang P Wang CT Bai J Jackson MB Chapman ER 《The Journal of biological chemistry》2003,278(47):47030-47037
The secretory vesicle protein synaptotagmin I (syt) plays a critical role in Ca2+-triggered exocytosis. Its cytoplasmic domain is composed of tandem C2 domains, C2A and C2B; each C2 domain binds Ca2+. Upon binding Ca2+, positively charged residues within the Ca2+-binding loops are thought to interact with negatively charged phospholipids in the target membrane to mediate docking of the cytoplasmic domain of syt onto lipid bilayers. The C2 domains of syt also interact with syntaxin and SNAP-25, two components of a conserved membrane fusion complex. Here, we have neutralized single positively charged residues at the membrane-binding interface of C2A (R233Q) and C2B (K366Q). Either of these mutations shifted the Ca2+ requirements for syt-liposome interactions from approximately 20 to approximately 40 microm Ca2+. Kinetic analysis revealed that the reduction in Ca2+-sensing activity was associated with a decrease in affinity for membranes. These mutations did not affect sytsyntaxin interactions but resulted in an approximately 50% loss in SNAP-25 binding activity, suggesting that these residues lie at an interface between membranes and SNAP-25. Expression of full-length versions of syt that harbored these mutations reduced the rate of exocytosis in PC12 cells. In both biochemical and functional assays, effects of the R233Q and K366Q mutations were not additive, indicating that mutations in one domain affect the activity of the adjacent domain. These findings indicate that the tandem C2 domains of syt cooperate with one another to trigger release via loop-mediated electrostatic interactions with effector molecules. 相似文献
11.
Calcium-dependent fusion of the plasma membrane fraction from human neutrophils with liposomes 总被引:1,自引:0,他引:1
J W Francis J E Smolen K J Balazovich R R Sandborg L A Boxer 《Biochimica et biophysica acta》1990,1025(1):1-9
A cell-free assay monitoring lipid mixing was used to investigate the role of Ca2+ in neutrophil membrane-liposome fusion. Micromolar concentrations of Ca2+ were found to directly stimulate fusion of inside-out neutrophil plasma membrane enriched fractions (from neutrophils subjected to nitrogen cavitation) with liposomes (phosphatidylethanolamine:phosphatidic acid, 4:1 molar ratio). In contrast, right-side-out plasma membranes and granule membranes did not fuse with liposomes in the presence of Ca2+. Similar results were obtained with two different lipid mixing assays. Fusion of the neutrophil plasma membrane-enriched fraction with liposomes was dependent upon the concentration of Ca2+, with threshold and 50% maximal rate of fusion occurring at 2 microM and 50 microM, respectively. Furthermore, the fusion was highly specific for Ca2+; other divalent cations such as Ba2+, Mg2+ and Sr2+ promoted fusion only at millimolar concentrations. Red blood cell (RBC) membranes were used in control studies. Ca2(+)-dependent fusion did not occur between right-side-out or inside-out RBC-vesicles and liposomes. However, if the RBC-vesicles were exposed to conditions which depleted spectrin (i.e., low salt), then Ca2(+)-dependent fusion was detected. Other quantitative differences between neutrophil and RBC membranes were found; fusion of liposomes with RBC membranes was most readily achieved with La3+ while neutrophil membrane-liposome fusion was most readily obtained with Ca2+. Furthermore, GTP gamma S was found to enhance Ca2(+)-dependent fusion between liposomes and neutrophil plasma membranes, but not RBC membranes. These studies show that plasma membranes (enriched fractions) from neutrophils are readily capable of fusing with artificial lipid membranes in the presence of micromolar concentrations of Ca2+. 相似文献
12.
Fusion of small unilamellar vesicles with viable EDTA-treated Escherichia coli cells. 总被引:1,自引:0,他引:1
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Fusion characteristics of EDTA-treated Escherichia coli cells with small unilamellar vesicles were investigated, using a membrane fusion assay based on resonance energy transfer. Ca2+-EDTA treatments of Escherichia coli O111:B4 (wild type), E. coli C600 (rough), and E. coli D21f2 (deep rough) which permeabilize the outer membrane by inducing the release of lipopolysaccharide and outer membrane proteins resulted in fusion activity of the intact and viable bacteria with small unilamellar vesicles. No fusion activity was observed when the EDTA treatment was omitted. Fusion could be elicited at low pH and by a combination of a higher pH and Ca2+. The low-pH-induced fusion was composed of a fast and a slow reaction. The latter and the Ca2+-induced fusion could be completely inhibited by trypsin treatments of the EDTA-treated cells, which also resulted in the simultaneous disappearance of two outer membrane protein bands (50 and 58 kilodaltons) and the appearance of proteins banding at 22, 52, and 54 kilodaltons. The most efficient fusion was obtained with negatively charged liposomes composed of cardiolipin. In contrast to the Ca2+-induced fusion, fusion was observed at low pH with small unilamellar vesicles containing lipids with decreased negative charge (phosphatidylserine). Fluorescent and phase-contrast microscopy revealed that essentially all bacteria were engaged in fusion. We propose that a Ca2+-EDTA treatment of E. coli cells results in the appearance of phospholipids and the exposure of a protein(s) in the outer leaflet of the outer membrane, both of which could mediate fusion with liposomes. 相似文献
13.
1. Virally mediated permeability changes are not brought about by neuraminidase or other enzyme. They are not mimicked by local anaesthetics such as benzyl alcohol or dibucaine. 2. Virus induces an increase in the exchange rate of membrane-bound Ca2+. The temperature-dependence, and sensitivity to concanavalin A, of the permeability change and of Ca2+ exchange are similar. 3. It is concluded that an increase in the exchangeability of membrane-bound Ca2+ constitutes an early and specific event after the attachment of Sendai virus to cultured cells. 相似文献
14.
Interaction of dextran sulfate with phospholipid surfaces and liposome aggregation and fusion 总被引:1,自引:1,他引:0
The binding of dextran sulfate to phospholipid liposomes was investigated by microelectrophoresis experiments. The polyanion binds to neutral phospholipid liposomes (DMPC and PE) only in the presence of Ca2+. If positively charged stearylamine is incorporated in the vesicles dextran sulfate is bound without Ca2+. Negatively charged phospholipids as PS do not bind dextran sulfate, even in the presence of millimolar concentrations of Ca2+. The adsorption of dextran sulfate results in an aggregation of vesicles due to a bridging mechanism. In all cases the aggregation is followed by a disaggregation toward higher dextran sulfate concentrations. The disaggregation process starts at polymer concentrations smaller than the concentration of the onset of saturation of the adsorption. By use of the probe dilution method a fusion of small DMPC and DMPC/PE vesicles in the presence of Ca2+ and dextran sulfate was found. 相似文献
15.
The ability of lipid asymmetry to regulate Ca(2+)-stimulated fusion between large unilamellar vesicles has been investigated. It is shown that for 100-nm-diameter LUVs composed of dioleoylphosphatidylcholine, dioleoylphosphatidylethanolamine, phosphatidylinositol, and dioleoylphosphatidic acid (DOPC/DOPE/PI/DOPA; 25:60:5:10) rapid and essentially complete fusion is observed by fluorescent resonance energy transfer techniques when Ca2+ (8 mM) is added. Alternatively, for LUVs with the same lipid composition but when DOPA was sequestered to the inner monolayer by incubation in the presence of a pH gradient (interior basic), little or no fusion is observed on addition of Ca2+. It is shown that the extent of Ca(2+)-induced fusion correlates with the amount of exterior DOPA. Further, it is shown that LUVs containing only 2.5 mol % DOPA, but where all the DOPA is in the outer monolayer, can be induced to fuse to the same extent and with the same rate as LUVs containing 5 mol % DOPA. These results strongly support a regulatory role for lipid asymmetry in membrane fusion and indicate that the fusogenic tendencies of lipid bilayers are largely determined by the properties of the monolayers proximate to the fusion interface. 相似文献
16.
The effects of local anesthetics on photosynthetic activity of pea chloroplasts were investigated in order to elucidate the role of Ca2+ in photosynthetic electron transport. Dibucaine, benzocaine and tetracaine were found to inhibit the O2-evolving activity. The inhibitory effect decreases in the order dibucaine greater than benzocaine greater than tetracaine greater than trimecaine similarly as does the potency to inhibit propagation of excitation in nerve fibre. As demonstrated in experiments with artificial donors and acceptors, the site of inhibition is the water-splitting site of PSII. The inhibitory power of the anesthetics grows with increasing ionic strength of the incubating mixture (by adding NaCl or MgCl2) and with pH; this is explained by occurrence of the neutral form of amine. At low concentrations the charged anesthetic acts as a protonofore; however, the inactivation of water splitting is not due to the protonophoric effect. The incubation is followed by the disappearance of ESR signal IIs. The role of Ca2+ and Ca2+-binding protein in PSII electron transport and its localization are discussed. 相似文献
17.
The lipid distribution in binary mixed membranes containing charged and uncharged lipids and the effect of Ca2+ and polylysine on the lipid organization was studied by the spin label technique. Dipalmitoyl phosphatidic acid was the charged, and spin labelled dipalmitoyl lecithin was the uncharged (zwitterionic) component. The ESR spectra were analyzed in terms of the spin exchange frequency, Wex. By measuring Wex as a function of the molar percentage of labelled lecithin a distinction between a random and a heterogeneous lipid distribution could be made. It is established that mixed lecithin-phosphatidic acid membranes exhibit lipid segregation (or a miscibility gap) in the fluid state. Comparative experiments with bilayer and monolayer membranes strongly suggest a lateral lipid segregation. At low lecithin concentration, aggregates containing between 25% and 40% lecithin are formed in the fluid phosphatidic acid membrane. This phase separation in membranes containing charged lipids is understandable on the basis of the Gouy-Chapman theory of electric double layers. In dipalmitoyl lecithin and in dimyristoyl phosphatidylethanolamine membranes the labelled lecithin is randomly distributed above the phase transition and has a coefficient of lateral diffusion of D = 2.8-10(-8) cm2/s at 59 degrees C. Addition of Ca2+ dramatically increases the extent of phase separation in lecithin-phosphatidic acid membranes. This chemically (and isothermally) induced phase separation is caused by the formation of crystalline patches of the Ca2+-bound phosphatidic acid. Lecithin is squeezed out from these patches of rigid lipid. The observed dependence of Wex on the Ca2+ concentration could be interpreted quantitatively on the basis of a two-cluster model. At low lecithin and Ca2+ concentration clusters containing about 30 mol % lecithin are formed. At high lecithin or Ca2+ concentrations a second type of precipitation containing 100% lecithin starts to form in addition. A one-to-one binding of divalent ions and phosphatidic acid at pH 9 was assumed. Such a one-to-one binding at pH 9 was established for the case of Mn2+ using ESR spectroscopy. Polylysine leads to the same strong increase in the lecithin segregation as Ca2+. The transition of the phosphatidic acid bound by the polypeptide is shifted from Tt = 47.5 degrees to Tt = 62 degrees C. This finding suggests the possibility of cooperative conformational changes in the lipid matrix and in the surface proteins in biological membranes. 相似文献
18.
K Nam S Kimura H Fujiki Y Imanishi 《Biochemical and biophysical research communications》1989,165(3):1256-1261
The effects of different types of lipid membrane defects on Ca2+-induced fusion of liposomes containing phosphatidylserine (PS) were investigated using fluorescent probes. Teleocidin enhanced the fusion of phospholipid vesicles in an assay system using terbium/dipicolinic acid during mixing of internal aqueous phases of vesicles upon fusion. 12-O-Tetradecanoylphorbol-13-acetate (TPA) suppressed the fusion. This latter phenomenon was also observed by measuring the excitation energy transfer. The promotion of membrane fusion by teleocidin was ascribed to dehydration of the membrane surface, the suppressive effect of TPA to desorption of Ca2+ from the membrane surface. Thus, Ca2+-induced fusion of PS vesicles was shown to be sensitive to defects of the membrane surface, but insensitive to defects of the hydrophobic core of the lipid membrane. 相似文献
19.
The effect of Ca2+ on a gel-to-liquid crystal transition as well as the mechanical properties of dipalmitoylphosphatidylcholine bilayers was studied by an ultrasonic technique. Transition temperature increased with increase in Ca2+ concentration, whereas the variation of ultrasonic anomalies indicated that dipalmitoylphosphatidylcholine bilayers exhibited maximum pseudocritical fluctuation at a Ca2+ concentration of about 10 mM. Hardening of dipalmitoylphosphatidylcholine membranes due to the Ca2+ binding was observed above 10 mM CaCl2, suggesting the lateral compression of the lipid bilayer by bound Ca2+. Long-range attraction between bound Ca2+ and the head groups of surrounding lipid molecules was proposed from these calcium effects. 相似文献
20.
Using an assay which allows continuous monitoring of the mixing of aqueous contents during membrane fusion, we have investigated the kinetics of calcium-phosphate-induced fusion of erythrocyte ghosts. In the presence of 10 mM phosphate, the threshold concentration for Ca2+-induced fusion was 1.25 mM, while the optimal concentration was approx. 1.75 mM Ca2+. Further enhancement of the cation concentration (greater than or equal to 2 mM) inhibited fusion of the ghosts. Initiation of fusion required the addition of phosphate prior to the addition of Ca2+, indicating that the combined interaction of Ca2+ and phosphate in or at the plane of the bilayer was a prerequisite for the induction of fusion. Furthermore, fusion was greatly facilitated upon transformation of calcium phosphate in the bulk medium from an amorphous to a solid, crystalline phase. It is suggested that membrane aggregation, and hence fusion, is facilitated by the formation of crystalline calcium phosphate nucleating on the ghost membrane. La3+, Mg2+ and Mn2+ did not trigger the fusion process, although aggregation of the ghosts did occur. Under conditions where calcium phosphate precipitation was inhibited, lanthanum phosphate precipitates facilitated fusion after prior treatment of ghosts with phosphate and Ca2+. These results indicated that fusion-prone conditions were induced prior to calcium phosphate precipitation. It is proposed that prior to calcium phosphate precipitation membrane changes are induced by separate interaction of Ca2+ and phosphate with the ghost membrane. Such an interaction could then render the ghosts susceptible to fusion and as soon as conditions are provided allowing close contact between adjacent membranes, fusion will be observed. 相似文献