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Interaction of the meso-tetrakis (4-N-methylpyridyl) porphyrin with gel and liquid state phospholipid vesicles

Sousa Neto, Diogenes de; Tabak, Marcel
Fonte: ACADEMIC PRESS INC ELSEVIER SCIENCE; SAN DIEGO Publicador: ACADEMIC PRESS INC ELSEVIER SCIENCE; SAN DIEGO
Tipo: Artigo de Revista Científica
ENG
Relevância na Pesquisa
58.005215%
The interaction of the cationic meso-tetrakis 4-N-methylpyridyl porphyrin (TMPyP) with large unilamellar vesicles (LUVs) was investigated in the present study. LUVs were formed by mixtures of the zwitterionic 1,2-dipalmitoyl-sn-glycero-phosphatidylcholine (DPPC) and anionic 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG) phospholipids, at different DPPG molar percentages. All investigations were carried out above (50 degrees C) and below (25 degrees C) the main phase transition temperature of the LUVs (similar to 41 degrees C). The binding constant values, K-b, estimated from the time-resolved fluorescence study, showed a significant increase of the porphyrin affinity at higher mol% DPPG. This affinity is markedly increased when the LUVs are in the liquid crystalline state. For both situations, the increase of the K-b value was also followed by a higher porphyrin fraction bound to the LUVs. The displacement of the vesicle-bound porphyrins toward the aqueous medium, upon titration with the salt potassium chloride (KCl), was also studied. Altogether, our steady-state and frequency-domain fluorescence quenching data results indicate that the TMPyP is preferentially located at the LUVs Stern layer. This is supported by the zeta potential studies...

End-to-end Distance Distribution in Fluorescent Derivatives of Bradykinin in Interaction with Lipid Vesicles

Montaldi, L. R.; Berardi, M.; Souza, E. S.; Juliano, L.; Ito, Amando Siuiti
Fonte: SPRINGER/PLENUM PUBLISHERS; NEW YORK Publicador: SPRINGER/PLENUM PUBLISHERS; NEW YORK
Tipo: Artigo de Revista Científica
ENG
Relevância na Pesquisa
57.88621%
Cellular membranes have relevant roles in processes related to proteases like human kallikreins and cathepsins. As enzyme and substrate may interact with cell membranes and associated co-factors, it is important to take into account the behavior of peptide substrates in the lipid environment. In this paper we report an study based on energy transfer in two bradykinin derived peptides labeled with the donor-acceptor pair Abz/Eddnp (ortho-aminobenzoic acid/N-[2,4-dinitrophenyl]-ethylenediamine). Time-resolved fluorescence experiments were performed in phosphate buffer and in the presence of large unilamelar vesicles of phospholipids, and of micelles of sodium dodecyl sulphate (SDS). The decay kinetics were analyzed using the program CONTIN to obtain end-to-end distance distribution functions f(r). Despite of the large difference in the number of residues the end-to-end distance of the longer peptide (9 amino acid residues) is only 20 % larger than the values obtained for the shorter peptide (5 amino acid residues). The proline residue, in position 4 of the bradykinin sequence promotes a turn in the longer peptide chain, shortening its end-to-end distance. The surfactant SDS has a strong disorganizing effect, substantially broadening the distance distributions...

Interação da porfirina catiônica meso-tetrakis (4-N-metilpiridil) com vesículas de fosfolipídio nos estados gel e líquido cristalino; Interaction of the cationic meso-tetrakis (4-N-methylpyridyl) porphyrin with gel and liquid state phospholipid vesicles

Sousa Neto, Diógenes de
Fonte: Biblioteca Digitais de Teses e Dissertações da USP Publicador: Biblioteca Digitais de Teses e Dissertações da USP
Tipo: Tese de Doutorado Formato: application/pdf
Publicado em 23/04/2014 PT
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68.55442%
Este estudo reúne os principais resultados de fluorescência estática e resolvida no tempo sobre a interação da porfirina meso-tetrakis (4-metilpiridil), na forma de base livre (TMPyP) e complexada com Zn2+ (ZnTMPyP), com vesículas de fosfolipídio. Adicionalmente foram utilizadas as técnicas de potencial zeta e espalhamento de luz dinâmico (DLS, do inglês "dynamic light scattering"). As vesículas de fosfolipídio foram formadas por dois conjuntos de fosfolipídios: saturados e insaturados. O primeiro grupo é formado pela mistura dos fosfolipídios zwiteriônico 1,2-dipalmitoil-sn-glicero-3-fosfocolina (DPPC) e aniônico 1,2-dipalmitoil-sn-3-glicero-[fosfo-rac-(1- glicerol)] (DPPG), a diferentes razões molares. Os estudos utilizando tais sistemas foram realizados abaixo (25oC) e acima (50oC) da temperatura de transição de fase gel-líquido cristalino destes fosfolipídios (~ 41oC). O segundo grupo é formado pela mistura dos fosfolipídios zwiteriônico 1-palmitoil-2-oleoil-sn-glicero-3-fosfocolina (POPC) e aniônico 1-palmitoil-2-oleoil-sn-glicero-3-fosfo(1-rac-glicerol) (POPG). Como a transição de fase destes dois fosfolipídios ocorre a temperaturas negativas, todos os experimentos foram realizados a 25oC (vesículas no estado líquido cristalino). Todos os sistemas foram preparados através do método de extrusão para a obtenção de vesículas grandes unilamelares (LUV...

Transfer of oleic acid between albumin and phospholipid vesicles.

Hamilton, J A; Cistola, D P
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /01/1986 EN
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48.315596%
The net transfer of oleic acid between egg phosphatidylcholine unilamellar vesicles and bovine serum albumin has been monitored by 13C NMR spectroscopy and 90% isotopically substituted [1-13C]oleic acid. The carboxyl chemical shifts of oleic acid bound to albumin were different from those for oleic acid in phospholipid vesicles. Therefore, in mixtures of donor particles (vesicles or albumin with oleic acid) and acceptor particles (fatty acid-free albumin or vesicles), the equilibrium distribution of oleic acid was determined from chemical shift and peak intensity data without separation of donor and acceptor particles. In a system containing equal masses of albumin and phospholipid and a stoichiometry of 4-5 mol of oleic acid per mol of albumin, the oleic acid distribution was pH dependent, with greater than or equal to 80% of the oleic acid associated with albumin at pH 7.4; association was greater than or equal to 90% at pH 8.0. Decreasing the pH below 7.4 markedly decreased the proportion of fatty acid bound to albumin; at pH 5.4, less than or equal to 10% of the oleic acid was bound to albumin and greater than 90% was associated with vesicles. The distribution was reversible with pH and was independent of whether vesicles or albumin acted as a donor. These data suggest that pH may strongly influence the partitioning of fatty acid between cellular membranes and albumin. The 13C NMR method is also advantageous because it provides information about the structural environments of oleic acid bound to albumin or phospholipid...

Formation and properties of 1000-A-diameter, single-bilayer phospholipid vesicles.

Enoch, H G; Strittmatter, P
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /01/1979 EN
Relevância na Pesquisa
48.284985%
Two methods are reported for the formation of large, uniform-sized phospholipid vesicles. The methods involve the treatment of phospholipid, in the form of either small, sonicated vesicles or a dry lipid film, at a molar ratio of deoxycholate to phospholipid of 1:2. Subsequent removal of deoxycholate yields a stable preparation of vesicles. These vesicles are bounded by a single bilayer, have an average diameter of 1000 A, and are readily separated from sonicated vesicles (230 A) by gel filtration on Sepharose 4B. Since the 1000-A vesicles are capable of trapping enzymes and other macromolecules, they may prove valuable for the delivery of liposome-entrapped solutes to cells and for the localization of peptide segments of a spectrum of membrane-bound proteins.

Binding of phospholipase C delta 1 to phospholipid vesicles.

Pawelczyk, T; Lowenstein, J M
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/05/1993 EN
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48.3744%
Binding of phospholipase C delta 1 (PLC delta) to phospholipid vesicles was studied using large, unilamellar phospholipid vesicles (LUVs). PLC delta bound weakly to vesicles composed of phosphatidylserine (PS) or phosphatidylcholine (PC) or phosphatidylethanolamine (PE) + PC, and even more weakly to vesicles composed of phosphatidylinositol. The enzyme bound strongly to LUVs composed of PE + PC and phosphatidylinositol 4,5-bisphosphate (PIP2) or sphingomyelin (SM). Binding of 50% of PLC delta occurred at 0.25 nmol/ml PIP2 when LUVs composed of PE + PC (molar ratio of 80:20), plus various amounts of PIP2, were used at a constant phospholipid concentration of 300 nmol/ml. When LUVs composed of PE + PC + PIP2 (molar ratio of 79:20:1) were tested as a function of increasing phospholipid concentration, 50% binding of PLC delta occurred at 1.2 nmol/ml PIP2 and 120 nmol/ml total phospholipid. Similar measurements were conducted with other phospholipids and PIP2 at a molar ratio of 99:1. These showed that 50% binding of PLC delta occurred at a level of 0.9 nmol/ml PIP2 with 80 nmol/ml PC; at 2.2 nmol/ml PIP2 with 170 nmol/ml PS; at 4.2 nmol/ml PIP2 with 320 nmol/ml PI; and at 0.26 nmol/ml PIP2 with 20 nmol/ml total liver phospholipids. Binding to phosphatidylinositol 4-phosphate was much weaker. When LUVs composed of PE + PC + SM (molar ratio 48:12:40) were tested as a function of increasing phospholipid concentration...

Fusion of phospholipid vesicles produced by the anti-tumour protein alpha-sarcin.

Gasset, M; Oñaderra, M; Thomas, P G; Gavilanes, J G
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/02/1990 EN
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48.245874%
The anti-tumour protein alpha-sarcin causes fusion of bilayers of phospholipid vesicles at neutral pH. This is demonstrated by measuring the decrease in the efficiency of the fluorescence energy transfer between N-(7-nitro-2-1,3-benzoxadiazol-4-yl)-dimyristoylphosphatidylethano lamine (NDB-PE) (donor) and N-(lissamine rhodamine B sulphonyl)-diacylphosphatidylethanolamine (Rh-PE) (acceptor) incorporated in dimyristoylphosphatidylcholine (DMPG) vesicles. The effect of alpha-sarcin is a maximum at 0.15 M ionic strength and is abolished at basic pH. alpha-Sarcin promotes fusion between 1,6-diphenylhexa-1,3,5-triene (DPH)-labelled DMPG and dipalmitoyl-PG (DPPG) vesicles, resulting in a single thermotropic transition for the population of fused phospholipid vesicles. Bilayers composed of DMPC and DMPG, at different molar ratios in the range 1:1 to 1:10 PC/PG, are also fused by alpha-sarcin. Freeze-fracture electron micrographs corroborate the occurrence of fusion induced by the protein. alpha-Sarcin also modifies the permeability of the bilayers, causing the leakage of calcein in dye-trapped PG vesicles. All of the observed effects reach saturation at a 50:1 phospholipid/protein molar ratio, which is coincident with the binding stoichiometry previously described.

Regulation of annexin I-dependent aggregation of phospholipid vesicles by protein kinase C.

Johnstone, S A; Hubaishy, I; Waisman, D M
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 15/09/1993 EN
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48.299487%
Annexin I is a member of the annexin family of Ca(2+)- and phospholipid-binding proteins. The ability of this protein to aggregate and to mediate the fusion of various types of vesicles has supported the hypothesis that this protein might be involved in intracellular membrane fusion processes such as exocytosis. Although annexin I has been described as a major in vitro substrate of both protein kinase C and the epidermal-growth-factor-receptor protein tyrosine kinase, the functional consequences of these phosphorylation events have not been investigated. In this paper we examine the effect of the phosphorylation of annexin I by protein kinase C on the phospholipid aggregation activity of the protein. The stoichiometry of phosphorylation of the protein was affected by the method of preparation of the phospholipid. Under optimal assay conditions protein kinase C catalysed the incorporation of 2.83 +/- 0.23 mol of phosphate/mol of annexin I (mean +/- S.E.M., n = 21). Studies with the Ca(2+)- and phospholipid-independent form of protein kinase C suggested that the phosphorylation of annexin I was stimulated by phospholipid in the absence of Ca2+, although maximal phosphorylation was achieved in the presence of both phospholipid and Ca2+. Phosphorylation of annexin I resulted in a dramatic decrease in the rate and extent of phospholipid vesicle aggregation...

Tryptophan fluorescence study on the interaction of pulmonary surfactant protein A with phospholipid vesicles.

Casals, C; Miguel, E; Perez-Gil, J
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 15/12/1993 EN
Relevância na Pesquisa
48.340205%
The fluorescence characteristics of surfactant protein A (SP-A) from porcine and human bronchoalveolar lavage were determined in the presence and absence of lipids. After excitation at either 275 or 295 nm, the fluorescence emission spectrum of both proteins was characterized by two maxima at about 326 and 337 nm, indicating heterogeneity in the emission of the two tryptophan residues of SP-A, and also revealing a partially buried character for these fluorophores. Interaction of both human and porcine SP-A with various phospholipid vesicles resulted in an increase in the fluorescence emission of tryptophan without any shift in the emission wavelength maxima. This change in intrinsic fluorescence was found to be more pronounced in the presence of dipalmitoyl phosphatidylcholine (DPPC) than with dipalmitoyl phosphatidylglycerol (DPPG), DPPC/DPPG (7:3, w/w) and 1-palmitoyl-sn-glycerol-3-phosphocholine (LPC). Intrinsic fluorescence of SP-A was almost completely unaffected in the presence of egg phosphatidylcholine (egg-PC). In addition, we demonstrated a shielding of the tryptophan fluorescence from quenching by acrylamide on interaction of porcine SP-A with DPPC, DPPG or LPC. This shielding was most pronounced in the presence of DPPC. In the case of human SP-A...

Study of the interaction between the antitumour protein alpha-sarcin and phospholipid vesicles.

Gasset, M; Martinez del Pozo, A; Oñaderra, M; Gavilanes, J G
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/03/1989 EN
Relevância na Pesquisa
48.514175%
alpha-Sarcin is a single polypeptide chain protein which exhibits antitumour activity by degrading the larger ribosomal RNA of tumour cells. We describe the interaction of a alpha-sarcin with lipid model systems. The protein specifically interacts with negatively-charged phospholipid vesicles, resulting in protein-lipid complexes which can be isolated by ultracentrifugation in a sucrose gradient. alpha-Sarcin causes aggregation of such vesicles. The extent of this interaction progressively decreases when the molar ratio of phosphatidylcholine increases in acidic vesicles. The kinetics of the vesicle aggregation induced by the protein have been measured. This process is dependent on the ratio of alpha-sarcin present in the protein-lipid system. A saturation plot is observed from phospholipid vesicles-protein titrations. The saturating protein/lipid molar ratio is 1:50. The effect produced by the antitumour protein on the lipid vesicles is dependent on neither the length nor the degree of unsaturation of the phospholipid acyl chain. However, the aggregation is dependent on temperature, being many times higher above the phase transition temperature of the corresponding phospholipid than below it. The effects of pH and ionic strength have also been considered. An increase in the ionic strength does not abolish the protein-lipid interaction. The effect of pH may be related to conformational changes of the protein. Binding experiments reveal a strong interaction between alpha-sarcin and acidic vesicles...

Incorporation into phospholipid vesicles of pore-like properties from Golgi membranes of lactating-rat mammary gland.

Wallace, A V; Kuhn, N J
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 15/05/1986 EN
Relevância na Pesquisa
48.364453%
The ability of rat mammary-gland Golgi membranes to produce monosaccharide-specific pores in phospholipid vesicles was investigated. The apparent ability of Triton X-100 extracts of Golgi membranes to form such pores was re-evaluated, since we have now found that an apparent pore is produced by the detergent alone. We therefore incorporated intact Golgi membranes (1 mg of protein) into egg-yolk phospholipid vesicles by direct sonication in the absence of any detergent. These vesicles retained about 0.6% of the total sucrose, but demonstrated selective permeability towards glucose compared with sucrose, with 19.8% of the glucose being lost during gel filtration on Sepharose 4B. This phenomenon seemed to be enhanced by the presence of acidic phospholipids and lysophosphatidylcholine, but was inhibited by inclusion of cholesterol in the vesicles. The best mixture of phospholipids comprised 6.5 mg of egg-yolk phospholipid, 1 mg of phosphatidylserine and 0.05 mg of lysophosphatidylcholine, where 32.9% of the glucose was lost. By using this optimum phospholipid mixture the pores were shown to be permeable to both glucose and mannitol, whereas sucrose and lactose were retained by the vesicles. Chaps (3- [(3-cholamidopropyl)dimethylammonio] propane-1-sulphonate)-solubilized membranes produced similar permeability in vesicles produced by dialysis of a solution of the phospholipids mixed with the membrane extract. This technique resulted in a greater loss of glucose...

Interaction of factor VIII-von Willebrand Factor with phospholipid vesicles.

Andersson, L O; Brown, J E
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 15/10/1981 EN
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48.20199%
The interaction of Factor VIII-von Willebrand Factor with phospholipid vesicles has been studied by using sucrose-density-gradient ultracentrifugation. When purified Factor VIII-von Willebrand Factor was run alone. Factor VIII activity and Factor VIIIR-Ag sedimented together to the lower half of the tube. Addition of phosphatidylserine/phosphatidylethanolamine vesicles at concentrations above 250 microgram/ml resulted in complete separation of Factor VIII activity and Factor VIIIR-Ag, the former appearing with the phospholipid on the top of the tube and the latter sedimenting as before. This separation was obtained even in the presence of proteinase inhibitors. Activation of Factor VIII-von Willebrand Factor by thrombin resulted in formation of a slow sedimenting component containing essentially all the Factor VIII activity, whereas the Factor VIIIR-Ag sedimented towards the bottom of the tube as before. The thrombin-induced Factor VIII activity was strongly bound to phospholipid vesicles as determined by density-gradient centrifugations at various Factor VIII concentrations and low concentrations of phospholipid. Based on certain assumptions a dissociation constant of 2.5 nM was calculated, a mechanism for the formation in vivo of the Factor X-activator complex is suggested.

A photophysical model for diphenylhexatriene fluorescence decay in solvents and in phospholipid vesicles.

Parasassi, T; De Stasio, G; Rusch, R M; Gratton, E
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /02/1991 EN
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48.331006%
The fluorescence decay of 1,6-diphenyl-1,3,5-hexatriene (DPH) in pure solvents and in phospholipid vesicles has been measured using frequency domain fluorometry. Data analysis uses a model with two energetically close excited states. The model explains the high quantum yield and the double exponential decay of DPH observed in some pure solvents and in phospholipid vesicles. This model assumes that after excitation to a first excited state, there is a rapid interconversion to a lower excited state and that most of the emission occurs from this state. The interconversion rates between the two excited states determine the average lifetime. For DPH in solvents, we find that the interconversion rates are solvent and temperature dependent. For DPH in phospholipid vesicles, we find that the back reaction rate from excited state 2 to excited state 1 (R12) is what determines the fluorescence properties. The phospholipid phase transition affects only this back reaction rate. The model was analyzed globally for a range of solvents, temperatures and vesicle composition. Of the six parameters of the model, only two, the interconversion rates between the two excited states, varied in all different samples examined. For DPH in phospholipid vesicles...

Electrical Properties of Phospholipid Vesicles

Schwan, H. P.; Takashima, S.; Miyamoto, V. K.; Stoeckenius, W.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /11/1970 EN
Relevância na Pesquisa
48.245874%
The capacitance of the membrane of phospholipid vesicles and the electrical properties of the vesicle interior have been determined. To this end the electrical properties of phospholipid vesicles have been investigated over a frequency range extending from 1 kHz to 100 MHz. The dielectric behavior is characterized by two dispersions, one placed between 1 kHz and 1 MHz and the other between 1 and 100 MHz. The relaxational behavior at low frequencies is explained by counterion movement tangential to the vesicle surface and a reasonable value for the fixed charge of the vesicles is calculated from the dispersion magnitude. The relaxation at high frequencies is of the Maxwell-Wagner type and appears caused by the phospholipid bilayer bounding the interior phase of the vesicles. It is consistent with the existence of a closed bilayer with a capacitance of about 2 μF/cm2 and an internal phase similar to the vesicle suspending medium. There is no indication of other than normally structured water inside the small vesicles.

Parameters affecting the fusion of unilamellar phospholipid vesicles with planar bilayer membranes

Fonte: The Rockefeller University Press Publicador: The Rockefeller University Press
Tipo: Artigo de Revista Científica
Publicado em 01/03/1984 EN
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48.198286%
It was previously shown (Cohen, F. S., J. Zimmerberg, and A. Finkelstein, 1980, J. Gen. Physiol., 75:251-270) that multilamellar phospholipid vesicles can fuse with decane-containing phospholipid bilayer membranes. An essential requirement for fusion was an osmotic gradient across the planar membrane, with the vesicle-containing (cis) side hyperosmotic with respect to the opposite (trans) side. We now report that unilamellar vesicles will fuse with "hydrocarbon-free" membranes subject to these same osmotic conditions. Thus the same conditions that apply to fusion of multilamellar vesicles with planar bilayer membranes also apply to fusion of unilamellar vesicles with these membranes, and hydrocarbon is not required for the fusion process. If the vesicles and/or planar membrane contain negatively charged lipids, divalent cation (approximately 15 mM Ca++) is required in the cis compartment (in addition to the osmotic gradient across the membrane) to obtain substantial fusion rates. On the other hand, vesicles made from uncharged lipids readily fuse with planar phosphatidylethanolamine planar membranes in the near absence of divalent cation with just an osmotic gradient. Vesicles fuse much more readily with phosphatidylethanolamine-containing than with phosphatidylcholine-containing planar membranes. Although hydrocarbon (decane) is not required in the planar membrane for fusion...

Interaction of the sperm adhesive protein, bindin, with phospholipid vesicles. II. Bindin induces the fusion of mixed-phase vesicles that contain phosphatidylcholine and phosphatidylserine in vitro

Fonte: The Rockefeller University Press Publicador: The Rockefeller University Press
Tipo: Artigo de Revista Científica
Publicado em 01/03/1985 EN
Relevância na Pesquisa
48.3224%
Bindin from sea urchin sperm associates with gel-phase phospholipid bilayers (Glabe, C. G., 1985, J. Cell Biol., 100:794-799). Bindin also interacts with phospholipid vesicles containing both gel-phase and fluid-phase domains and thereby induces their aggregation. Association of bindin with vesicles containing gel-phase domains of dipalmitoylphosphatidylcholine (DPPC) and fluid-phase domains of brain phosphatidylserine (PS) was found to result in the fusion of the vesicles. After incubation with bindin, these mixed-phase vesicles were much larger as determined by gel filtration chromatography and electron microscopic observations of negatively stained samples. The average diameter of the vesicles after incubation was 190 +/- 109 nm compared with 39 +/- 20 nm for vesicles incubated in the absence of bindin. Resonance energy transfer studies also indicated that bindin induces the fusion of vesicle bilayers. Two fluorescent probes (NBD-PE and Rh- PE) were incorporated into the membrane of mixed-phase DPPC:PS vesicles at a density of 0.5 mol%, where efficient energy transfer occurs between the probes. The efficiency of energy transfer was proportional to the concentration of the fluorescence energy acceptor in the bilayer. The fluorescent vesicles were mixed with an excess of unlabeled target vesicles to quantify fusion. After bindin addition...

Fusion of phospholipid vesicles with planar phospholipid bilayer membranes. II. Incorporation of a vesicular membrane marker into the planar membrane

Fonte: The Rockefeller University Press Publicador: The Rockefeller University Press
Tipo: Artigo de Revista Científica
Publicado em 01/03/1980 EN
Relevância na Pesquisa
48.20199%
Fusion of multilamellar phospholipid vesicles with planar phospholipid bilayer membranes was monitored by the rate of appearance in the planar membrane of an intrinsic membrane protein present in the vesicle membranes. An essential requirement for fusion is an osmotic gradient across the planar membrane, with the cis side (the side containing the vesicles) hyperosmotic to the opposite (trans) side; for substantial fusion rates, divalent cation must also be present on the cis side. Thus, the low fusion rates obtained with 100 mM excess glucose in the cis compartment are enhanced orders of magnitude by the addition of 5- 10 mM CaCl2 to the cis compartment. Conversely, the rapid fusion rates induced by 40 mM CaCl2 in the cis compartment are completely suppressed when the osmotic gradient (created by the 40 mM CaCl2) is abolished by addition of an equivalent amount of either CaCl2, NaCl, urea, or glucose to the trans compartment. We propose that fusion occurs by the osmotic swelling of vesicles in contact with the planar membrane, with subsequent rupture of the vesicular and planar membranes in the region of contact. Divalent cations catalyze this process by increasing the frequency and duration of vesicle-planar membrane contact. We argue that essentially this same osmotic mechanism drives biological fusion processes...

Fusion of phospholipid vesicles with planar phospholipid bilayer membranes. I. Discharge of vesicular contents across the planar membrane

Fonte: The Rockefeller University Press Publicador: The Rockefeller University Press
Tipo: Artigo de Revista Científica
Publicado em 01/03/1980 EN
Relevância na Pesquisa
48.194146%
Multilamellar phospholipid vesicles are introduced into the cis compartment on one side of a planar phospholipid bilayer membrane. The vesicles contain a water-soluble fluorescent dye trapped in the aqueous phases between the lamellae. If a vesicle containing n lamellae fuses with a planar membrane, an n-1 lamellar vesicle should be discharged into the opposite trans compartment, where it would appear as a discernible fluorescent particle. Thus, fusion events can be assayed by counting the number of fluorescent particles appearing in the trans compartment. In the absence of divalent cation, fusion does not occur, even after vesicles have been in the cis compartment for 40 min. When CaCl2 is introduced into the cis compartment to a concentration of greater than or equal to 20 mM, fusion occurs within the next 20 min; it generally ceases thereafter because of vesicle aggregation in the cis compartment. With approximately 3 x 10(8) vesicles/cm3 in the cis compartment, about 25-50 fusion events occur following CaCl2 addition. The discharge of vesicular contents across the planar membrane is the most convincing evidence of vesicle-membrane fusion and serves as a model for that ubiquitous biological phenomenon--exocytosis.

Kinetics of vitamin D3 metabolism by cytochrome P450scc (CYP11A1) in phospholipid vesicles and cyclodextrin

Tuckey, Robert C.; Nguyen, Minh N.; Slominski, Andrzej
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
EN
Relevância na Pesquisa
48.47706%
Vitamin D3 can be hydroxylated sequentially by cytochrome P450scc (CYP11A1) producing 20-hydroxyvitamin D3, 20,23-dihydroxyvitamin D3 and 17,20,23-trihydroxyvitamin D3. The aim of this study was to characterize the ability of vitamin D3 to associate with phospholipid vesicles and to determine the kinetics of metabolism of vitamin D3 by P450scc in vesicles and in 2-hydroxypropyl-β-cyclodextrin (cyclodextrin). Gel filtration of phospholipid vesicles showed that the vitamin D3 remained quantitatively associated with the phospholipid membrane. Vitamin D3 exchanged between vesicles at a rate 3.8-fold higher than for cholesterol exchange and was stimulated by N-62 StAR protein. The Km of P450scc for vitamin D3 in vesicles was 3.3 mol vitamin D3/mol phospholipid and the rate of conversion of vitamin D3 to 20-hydroxyvitamin D3 was first order with respect to the vitamin D3 concentration for the range of concentrations of vitamin D3 that could be incorporated into the vesicle membrane. 20-Hydroxyvitamin D3 was further hydroxylated by P450scc in vesicles, producing primarily 20,23-dihydroxyvitamin D3, with Km and kcat values 22- and 6-fold lower than those for vitamin D3, respectively. 20,23-Dihydroxyvitamin D3 was converted to 17,20,23-trihydroxyvitamin D3 with even lower Km and kcat values. Vitamin D3 and cholesterol were metabolized with comparable efficiencies in cyclodextrin...

Interaction between ion channel-inactivating peptides and anionic phospholipid vesicles as model targets.

Encinar, J A; Fernandez, A M; Gavilanes, F; Albar, J P; Ferragut, J A; Gonzalez-Ros, J M
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /09/1996 EN
Relevância na Pesquisa
48.194146%
Studies of rapid (N-type) inactivation induced by different synthetic inactivating peptides in several voltage-dependent cation channels have concluded that the channel inactivation "entrance" (or "receptor" site for the inactivating peptide) consists of a hydrophobic vestibule within the internal mouth of the channel, separated from the cytoplasm by a region with a negative surface potential. These protein domains are conformed from alternative sequences in the different channels and thus are relatively unrestricted in terms of primary structure. We are reporting here on the interaction between the inactivating peptide of the Shaker B K+ channel (ShB peptide) or the noninactivating ShB-L7E mutant with anionic phospholipid vesicles, a model target that, as the channel's inactivation "entrance," contains a hydrophobic domain (the vesicle bilayer) separated from the aqueous media by a negatively charged vesicle surface. When challenged by the anionic phospholipid vesicles, the inactivating ShB peptide 1) binds to the vesicle surface with a relatively high affinity, 2) readily adopts a strongly hydrogen-bonded beta-structure, likely an intramolecular beta "hairpin," and 3) becomes inserted into the hydrophobic bilayer by its folded N-terminal portion...