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W L Vaz

Publications and source records attributed to W L Vaz.

At least 37 records · Page 2Linked to original sources
PubMed1987

A 31P-NMR study on multilamellar liposomes formed from the lipids of a thermophilic bacterium.

The membrane lipids of a thermophilic bacterium, Thermus SPS11, isolated from thermal springs in São Pedro do Sul, Portugal, were fractionated by chromatography on silica gel. The total lipid extract was found to contain one major phospholipid (PL), which accounts for about 90% of the total lipid phosphorous, and one major glycolipid (GL), which accounts for about 95% of the total carbohydrate in the non-phospholipid fraction. The membranes also contain about 11% by weight of a complex mixture of carotenoids (CA). Multilamellar liposomes, in excess water, formed from PL and mixtures of PL with GL and CA in proportions found in the natural membrane were investigated by proton-decoupled 31P-nuclear magnetic resonance (NMR) spectroscopy and X-ray diffraction. All mixtures examined were found to be in a lamellar phase with disordered hydrophobic chains with no evidence for "non-bilayer structures" between 23 degrees and 85 degrees C. Compared to bilayers formed from pure PL or mixtures of PL and CA, significantly larger values for the chemical shift anisotropy of the 31P-NMR powder patterns were obtained from bilayers formed from mixtures of PL and GL, at temperatures above 75 degrees C, and mixtures of PL, GL and CA at all temperatures examined. These differences are interpreted in terms of changes in the order of the bilayer and/or changes in the orientation of the phosphate moiety of PL. The significance of these results to the thermophily of the bacterium is discussed.

Liposomes↗
PubMed1986

Effect of short-chain primary alcohols on fluidity and activity of sarcoplasmic reticulum membranes.

Intramolecular excimer formation with the fluorescent probe 1,3-di(1-pyrenyl)propane, differential scanning calorimetry, and X-ray diffraction were used to assess the effect of ethanol, 1-butanol, and 1-hexanol on the bilayer organization in model membranes, sarcoplasmic reticulum (SR) lipids and native SR membranes. These alcohols have fluidizing effects on membranes and lower the main transition temperature of dimyristoylphosphatidylcholine (DMPC), but only 1-hexanol alters the cooperativity of the phase transition and significantly increases the thickness of DMPC bilayers. The interaction of the three alcohols with the SR Ca2+ pump was also investigated. Hydrolysis of ATP and coupled Ca2+ uptake are differently sensitive to the three alcohols. Whereas ethanol and 1-butanol inhibited the Ca2+ uptake, 1-hexanol stimulated it. Nevertheless, the energetic efficiency of the pump (Ca2+/ATP) is not significantly affected by ethanol or 1-hexanol, but uncoupling was observed with 1-butanol at high concentrations. The different effects of alcohols on the activity of SR membranes rule out an unitary mechanism of action on the basis of fluidity changes induced in the lipid bilayer. Depending on the chain length, the alcohols interact with the SR membranes in different domains, perturbing differently the Ca2+-pump activity.

Alcohols↗
PubMed1986

Determination of the critical micelle concentration of surfactants using the fluorescent probe N-phenyl-1-naphthylamine.

The fluorescence quantum yield of N-phenyl-1-naphthylamine (NPN) increases about 10-fold and the wavelength of maximum fluorescence emission is blue-shifted when this molecule partitions into the apolar core of micellar structures from the aqueous phase. This property allowed the utilization of NPN as a fluorescent indicator of micelle formation by 14 different surfactants belonging to the families of alkyltrimethylammonium halides, alkylsulfates, alkylbetaines, alkylglucosides, and bile salts. The critical micelle concentrations (CMCs) determined with NPN agreed well with literature values. In this work NPN was used at a concentration of 10(-6) M which allowed determination of CMCs in the range between approximately 10(-5) and greater than 10(-2) M. With high-sensitivity instrumentation considerably lower NPN concentrations can be used and consequently considerably lower CMCs can be rapidly and accurately determined.

1-Naphthylamine↗
PubMed1985

An X-ray diffraction and differential scanning calorimetric study on the effect of sucrose on the properties of phosphatidylcholine bilayers.

The influence of sucrose, between 0 and 70% in the aqueous phase, upon multilamellar liposomes of dimyristoylphosphatidylcholine was examined by differential scanning calorimetry and X-ray diffraction analysis. Increasing concentrations of sucrose increase the temperatures of both the main transition and the pretransition of the lipid. The effect is greater on the pretransition than on the main transition. At 35 degrees C the interlamellar spacing in the multilamellar liposomes is reduced by increasing sucrose concentration in the aqueous phase and no significant effects are seen in the chain lattice of the bilayers. This result is interpreted as a dehydrating effect of sucrose upon the bilayer-water system at 35 degrees C. At 5 degrees C the interlamellar spacing is increased and this increase is, at high (70%) sucrose concentrations, attributable to an untilting of the lipid acyl chains with no change in the thickness of the aqueous layers in the multilamellae.

Calorimetry, Differential Scanning↗
PubMed1985

Translational diffusion of lipids in liquid crystalline phase phosphatidylcholine multibilayers. A comparison of experiment with theory.

A systematic study of the translational diffusion of the phospholipid derivative N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine (NBD-PE) has been undertaken in liquid crystalline phase phosphatidylcholine bilayers by using the fluorescence recovery after photobleaching technique. This work was done with the intention of comparing the experimental results with the predictions of theoretical models for diffusion in membranes. The following is shown. For NBD-PE, the dependence of the translational diffusion coefficient (Dt) upon the acyl chain length of the diffusant is not that predicted by continuum fluid hydrodynamic models for diffusion in membranes [Saffman, P.G., & Delbrueck, M. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 3111-3113; Hughes, B. D., Pailthorpe, B. A., & White, L. R. (1981) J. Fluid Mech. 110, 349-372]. Plots of Dt vs. 1/T (Arrhenius plots) are nonlinear in dilauroyl-phosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) bilayers where the acyl chain composition of the NBD-PE is matched with that of the host bilayer lipid. This suggests that a "free volume" model may be appropriate for the description of lipid diffusion in lipid bilayers. In bilayers of phosphatidylcholines with saturated acyl chains at the same "reduced temperature", the magnitude of Dt follows the order distearoylphosphatidylcholine greater than DPPC greater than DMPC greater than DLPC. This is the inverse of what may be expected from the hydrodynamic model but is in agreement with the free volume in these bilayers.(ABSTRACT TRUNCATED AT 250 WORDS)

Diffusion↗
PubMed1985

A comparison of the translational diffusion of a normal and a membrane-spanning lipid in L alpha phase 1-palmitoyl-2-oleoylphosphatidylcholine bilayers.

We have used the fluorescence recovery after photobleaching technique to study the translational diffusion, in L alpha phase multibilayers of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), of fluorescent derivatives of 1-palmitoyl-2-oleoylphosphatidylethanolamine (NBD-POPE) and a membrane-spanning phosphatidylethanolamine (NBD-MSPE). The latter derivative was prepared from a membrane-spanning glycerol-dialkyl-glycerol tetraether lipid isolated from the thermophilic and acidophilic archaebacterium Sulfolobus solfataricus. The translational diffusion was examined between about 15 degrees and 45 degrees C. It is shown that over this temperature range the translational diffusion coefficient for NBD-MSPE is approximately 2/3 that for NBD-POPE which spans only one monolayer of the bilayer. The result is interpreted in terms of existing models for translational diffusion in lipid membranes.

Archaea↗
PubMed1984

Modulation of sarcoplasmic reticulum Ca2+-pump activity by membrane fluidity.

Intramolecular excimerization of 1,3-di-1-pyrenylpropane [Py(3)Py] was used to assess the fluidity of sarcoplasmic reticulum membranes (SR); on the basis of the spectral data, the probe incorporates completely inside the membrane probably somewhere close to the polar head groups of phospholipid molecules, however not in the very hydrophobic core. The excimerization rate is very sensitive to lipid phase transitions, as revealed by thermal profiles of dimyristoyl-phosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC) bilayers. Cholesterol abolishes pretransitions and broadens the thermal profiles of the main transitions which vanish completely at 50 mol % sterol. Excimer formation in liposomes of SR total lipid extracts does not show any sharp transitions, as in the case of DMPC and DPPC. However, the plots display discontinuities at about 20 degrees C which are broadened by cholesterol and not observed at 50 mol % sterol. Also cholesterol has been incorporated in native SR membranes by an exchange technique allowing progressive enrichment without changing the phospholipid/protein molar ratio. As in liposomes, discontinuities of excimer formation at 20 degrees C are broadened by cholesterol enrichment. The full activity of uncoupled Ca2+-ATPase is only affected by cholesterol above a molar ratio to phospholipid of 0.4. However, a significant decrease in activity (about 20%) is only noticed at a ratio of 0.6 (the highest technically achieved); at this ratio, about 28 lipid molecules per Ca2+-ATPase are expected to be relatively free from cholesterol interaction. The vesicle structure is still intact at this high ratio, as judged from the absence of basal activity (not Ca2+ stimulated).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1983

Distribution and mobility of murine histocompatibility H-2Kk antigen in the cytoplasmic membrane.

The topographical distributions and mobilities of the murine histocompatibility antigen H-2Kk and of concanavalin A (Con A) binding sites have been studied on a murine lymphoma cell line. The spatial distribution of H-2Kk antigens, the average distance between H-2Kk antigens and Con A binding sites, and the separation of different determinants on the H-2Kk antigen itself were determined by using fluorescence resonance energy-transfer measurements with a dual-laser flow sorter. From the lack of energy transfer between bound monoclonal anti-H-2Kk antibodies conjugated with fluorescein (donor) and rhodamine (acceptor), we conclude that the H-2Kk antigen exists without appreciable clustering on the cell surface. Substantial energy transfer between appropriately labeled Con A and antibodies bound to the H-2Kk antigen shows that the two populations are interspersed. Donor/acceptor pairs of monoclonal antibodies binding to different determinants on the same H-2Kk antigen exhibited a degree of energy transfer indicative of a mean separation of 8.6 nm between the sites. Time-resolved phosphorescence anisotropy measurements with anti-H-2Kk antibodies labeled with eosin or erythrosin yielded rotational mobility information for the antigen-antibody complexes on the cell membrane. The rotational correlation time of 10-20 mus and the finite residual anisotropy are compatible with an uniaxial mode of rotation of monomeric antigen around its transmembrane portion and, thus, provide additional evidence for an unclustered distribution. Capping by rabbit anti-mouse IgG immobilized the antigen-antibody complex. Fluorescence recovery after photobleaching was used to calculate an apparent lateral diffusion coefficient of 5 +/- 3 X 10(-10) cm2 . s-1 for the H-2Kk antigen labeled with fluoresceinated IgG or its corresponding Fab fragment.

Animals↗
PubMed1982

Fluidity of sarcoplasmic reticulum membranes investigated with dipyrenylpropane, an intramolecular excimer probe.

Intramolecular excimer formation with 1,3-di(1-pyrenyl)propane was used to probe the fluidity of sarcoplasmic reticulum (SR) membranes where the probe could be incorporated (in a probe/lipid molar ratio of 1/2000 or less) without inducing any detectable damage. The temperature was varied between -1 and 40 degrees C. The fluidity of the native SR membrane changes from 52 cP at 40 degrees C to 325 cP at 5 degrees C. The native SR membranes are less fluid than liposomes prepared from total membrane lipids. The fluidity of membranes reconstituted from the SR ATPase and a total lipid extract of the SR depends on the lipid/protein molar ratio and sharply decreases when this ratio becomes lower than 44, for all temperatures studied. This is in accord with literature reports describing the occurrence of approximately 30 lipid molecules around the Ca-ATPase molecule in the SR membrane. Arrhenius plots of the excimer to monomer fluorescence intensity ratio in native and reconstituted SR membranes display a break at about 20 degrees C which is not observed when the lipid/protein ratio is lower than 44. This break is interpreted as being a characteristic of the lipid portion of the membrane.

Fluorescent Dyes↗
PubMed1982

Size dependence of the translational diffusion of large integral membrane proteins in liquid-crystalline phase lipid bilayers. A study using fluorescence recovery after photobleaching.

The translational diffusion of bovine rhodopsin, the Ca2+-activated adenosinetriphosphatase of rabbit muscle sarcoplasmic reticulum, and the acetylcholine receptor monomer of Torpedo marmorata has been examined at a high dilution (molar ratios of lipid/protein greater than or equal to 3000/1) in liquid-crystalline phase phospholipid bilayer membranes by using the fluorescence recovery after photobleaching technique. These integral membrane proteins having molecular weights of about 37 000 for rhodopsin, about 100 000 for the adenosinetriphosphatase, and about 250 000 for the acetylcholine receptor were reconstituted into membranes of dimyristoylphosphatidylcholine (rhodopsin and acetylcholine receptor), soybean lipids (acetylcholine receptor), and a total lipid extract of rabbit muscle sarcoplasmic reticulum (adenosinetriphosphatase). The translational diffusion coefficients of all the proteins at 310 K were found to be in the range (1-3) X 10(-8) cm2/s. In consideration of the sizes of the membrane-bound portions of these proteins, this result is in agreement with the weak dependence of the translational diffusion coefficient upon diffusing particle size predicted by continuum fluid hydrodynamic models for the diffusion in membranes [Saffman, P. G., & Delbrück, M. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 3111-3113]. Lipid diffusion was also examined in th same lipid bilayers with the fluorescent lipid derivative N-(7-nitro-2,1,3-benzoxadiazol-4-yl)dimyristoylphosphatidylethanolamine. The translational diffusion coefficient for this lipid derivative was found to be in the range (9-14) X 10(-8) cm2/s at 310 K. In consideration of the dimensions of the lipid molecule, this value for the lipid diffusion coefficient is in agreement with the continuum fluid hydrodynamic model only if a near-complete slip boundary condition is assumed at the bilayer midplane. Alternatively, kinetic diffusion models [Träuble, H., & Sackmann. E. (1972) J. Am. Chem. Soc. 94, 4499-4510] may have to be invoked to explain the lipid diffusion behavior.

Acetylcholine↗
PubMed1982

Investigation of human erythrocyte ghost membranes with intramolecular excimer probes.

Human erythrocyte ghost membranes have been investigated using two intramolecular excimer probes, di(1-pyrenyl)propane and di(1-pyrenylmethyl) ether. Values for the viscosity of the direct probe environment in the ghost membranes range from 76 cP at 37 degrees C to 570 cP at 5 degrees C, as reported for di(1-pyrenyl(propane, with liquid paraffin as the reference solvent. For the activation energy of the excimer formation process, determined here mainly by the viscosity of the medium, a value of 37 kJ/mol is obtained. The other probe molecule reports a higher local viscosity, 133 cP at 37 degrees C, as well as a higher activation energy of excimer formation, 54 kJ/mol. Neither thermotropic phase transitions nor temperature hysteresis effects are observed within the temperature range (0 to 40 degrees C) studied. From the vibrational structure of the fluorescence spectrum of di(1-pyrenylmethyl) ether, a polarity of the probe environment close to that of hexanol (epsilon - 13.3) results for the erythrocyte ghost membranes. The polarity measured in egg phosphatidylcholine membranes and in multibilayers of dimyristoylphosphatidylcholine is slightly larger, comparable to that of butanol (epsilon = 17.5), whereas a polarity comparable to that of methanol (epsilon = 32.7) is observed for aqueous micellar solutions of sodium dodecyl sulphate. Further, from the wavelength shifts in the absorption spectrum of di(1-pyrenyl)propane and di(1-pyrenylmethyl) ether, the polarizability of the probe surroundings can be determined, leading to a surprisingly high value for the apparent refractive index. This is attributed to a high local density of the direct environment of the probe, for which a location between the membrane/water interface and the unpolar bilayer mid-plane is deduced.

Erythrocyte Membrane↗
PubMed1981

Translational mobility of glycophorin in bilayer membranes of dimyristoylphosphatidylcholine.

The translational diffusion of the integral membrane sialoglycoprotein from erythrocyte membranes, glycophorin, incorporated into bilayer membranes of dimyristoyl-phosphatidylcholine at a protein/lipid molar ratio of 1:4500 was examined by using the fluorescence redistribution after photobleaching technique. A plot of the diffusion coefficient vs. temperature shows a sharp decrease in the rate of diffusion at about 15 degrees C. This sharp diffusion transition is at a temperature some 9 degrees C lower than the calorimetrically measured lipid gel-liquid crystalline phase transition temperature of the system. The difference between the diffusion transition temperature and the lipid phase transition temperature is attributed toi a localized fluidizing effect of the protein upon the gel phase lipid. The value of the diffusion coefficient above 15 degrees C was found to be (1-2) x 10(-8) cm(2)s(-1), and below 15 degrees C it was lower than about 5 x 10(-11)cm(2)s(-1). The fluorescence recovery in the bleached area as a consequence of diffusional redistribution appeared to be due to a single diffusing species at temperatures above 15 degrees C and due to more than one diffusing species below this temperature.

Dimyristoylphosphatidylcholine↗
PubMed1979

Interactions of La3+ with phosphatidylserine vesicles. Binding, phase transition, leakage and fusion.

The interaction of La3+ with phosphatidylserine vesicles is elucidated by binding studies, differential scanning calorimetry, X-ray diffraction, freeze fracture electron microscopy, and release of vesicle contents. La3+ effectively competes with Ca2+ for phosphatidylserine binding sites. The saturation level is close to a La/lipid ratio of 1:3. A concentration of 0.1 mM of La3+ is sufficient to induce fusion between sonicated vesicles.

Calorimetry, Differential Scanning↗
PubMed1979

The rotational diffusion of cytochrome b5 in lipid bilayer membranes. Influence of the lipid physical state.

A derivative of the integral membranes protein, cytochrome b5, has been prepared in which the native heme group has been replaced by the structurally similar rhodium(III)-protoporphyrin IX. This metalloporphyrin has a finite triplet yield with a single exponential decay time of 22 microsecond in water. After insertion of the metalloporphyrin into the protein, its triplet-state decay becomes strongly nonexponential with at least three equal amplitude components with time constants varying over a range of 100. The derivatized protein has been incorporated into unilamellar liposomes prepared from dimyristoyllecithin, and the rotational diffusion of the protein in the lipid bilayer has been studied at temperatures above and below the lipid phase transition temperature via triplet absorbance anisotropy decay. The anisotropy decay curves are biphasic both above and below the lipid phase transition. The rotational diffusion constant is found to be 2.4 X 10(5) s-1 at 35 degrees C, and 1.1 X 10(4) s-1 at 10 degrees C, both being calculated from the fast decay component. The ratio of the limiting anisotropy to the initial anisotropy is 0.6 at both temperatures. This implies a cone of restricted motion of 34 degrees for the protein in the bilayer.

Animals↗
PubMed1979

Lateral mobility of an amphipathic apolipoprotein, ApoC-III, bound to phosphatidylcholine bilayers with and without cholesterol.

The technique of fluorescence recovery after photobleaching was used to investigate the lateral mobility of a fluorescein-labeled amphipathic apolipoprotein, ApoC-III, bound to multibilayers prepared from dipalmitoyl phosphatidylcholine, egg phosphatidylcholine, and a 1:1 (molar ratio) mixture of egg phosphatidylcholine and cholesterol. In dipalmitoyl phosphatidylcholine bilayers the lateral diffusion coefficient (D) for the protein is about 2 x 10(-9) cm(2) sec(-1) at 20 degrees C and about 9 x 10(-8) cm(2) sec(-1) at 45 degrees C. Plots of D versus temperature in this system show a transition between about 30 and 35 degrees C. Arrhenius activation energies for the diffusion in this case between 15 and 30 degrees C and between 35 and 45 degrees C are 28.5 and 7.0 kcal mol(-1), respectively (1 calorie = 4.18 joules). In egg phosphatidylcholine bilayers, D is about 3 x 10(-8) cm(2) sec(-1) at 20 degrees C and the Arrhenius activation energy for diffusion is 8.1 kcal mol(-1) between 15 and 35 degrees C in this system. In bilayers prepared from an equimolar mixture of egg phosphatidylcholine and cholesterol D at 20 degrees C is about 1.4 x 10(-9) cm(2) sec(-1) and the Arrhenius activation energy for the diffusion of the protein in this system between 15 and 35 degrees C is 15.1 kcal mol(-1). Light-scattering and fluorescence-polarization results indicate that binding of this protein does not affect the gel-to-liquid crystalline phase transition of bilayer membranes but does mediate a major, reversible aggregation of the vesicles at about 33 degrees C. These results lend support to the view that ApoC-III resides in the head-group region of the bilayer and suggest that its lateral diffusion coefficient represents an upper bound for integral membrane proteins.

Apolipoproteins↗