Biomedical subjects

T E Thompson

Publications and source records attributed to T E Thompson.

At least 73 records · Page 4Linked to original sources

PubMed1986

Physical and chemical modifications of adriamycin:iron complex by phospholipid bilayers.

Adriamycin (ADM) and the ADM:Fe(III) complex both interact with phosphatidylcholine bilayers in aqueous vesicle dispersions. The immediate interaction of either ADM or ADM:Fe(III) with phospholipid causes little change in their absorption or emission spectra, but considerably increases the steady-state fluorescence anisotropy of both species. This is followed by a conversion of the ADM:Fe(III) complex (but not of metal-free ADM) into a new compound in periods ranging from minutes to hours depending upon the Fe(III) concentration. This reaction does not require the presence of unsaturated acyl chains, net negatively charged phospholipid head groups, or the participation of molecular oxygen. This new compound has a characteristic absorption and fluorescence emission spectra which differ from those of the ADM:Fe(III) or of metal-free ADM. It can be isolated from the aqueous lipid dispersion by Folch extraction under acidic conditions. It is very lipophilic in comparison to ADM or the ADM:Fe(III) complex. It may be similar to the compound reported to form between cardiolipin and Adriamycin. Preliminary results indicate that it also forms spontaneously in intact biological membranes. Its highly lipophilic character may confine it to bilayers and membranes.

Doxorubicin↗

PubMed1985

Two types of hydrocarbon chain interdigitation in sphingomyelin bilayers.

Vibrational Raman spectroscopic experiments have been performed as a function of temperature on aqueous dispersions of synthetic DL-erythro-N-lignoceroylsphingosylphosphocholine [C(24):SPM], a racemic mixture of two highly asymmetric hydrocarbon chain length sphingomyelins. Raman spectral peak-height intensity ratios of vibrational transitions in the C-H stretching-mode region show that the C(24):SPM-H2O system undergoes two thermal phase transitions centered at 48.5 and 54.5 degrees C. Vibrational data for fully hydrated C(24):SPM are compared to those of highly asymmetric phosphatidylcholine dispersions. The Raman data are consistent with the plausible model that the lower temperature transition can be ascribed to the conversion of a mixed interdigitated gel state (gel II) to a partially interdigitated gel state (gel I) and that the higher temperature transition corresponds to a gel I----liquid-crystalline phase transition. The observation of a mixed interdigitated gel state (gel II) at temperatures below 48.5 degrees C implies that biological membranes may have lipid domains in which some of the lipid hydrocarbon chains penetrate completely across the entire hydrocarbon width of the lipid bilayer.

Kinetics↗

PubMed1985

Properties of a specific glycolipid transfer protein from bovine brain.

A transfer protein specific for glycolipids has been isolated from bovine brain. As judged by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, the protein is 68% pure and has a molecular weight of 20 000. Three different assays were employed to study the protein's specificity and glycolipid binding properties. The protein transferred several different neutral glycosphingolipids and ganglioside GM1 equally well, but failed to accelerate phosphatidylcholine or sphingomyelin intervesicular movement. The protein's ability to interact with glycolipids was strongly influenced by the physical properties of the matrix phospholipid in which the glycolipids reside. Both the phase state of the phospholipid matrix and bilayer curvature affected glycolipid intervesicular transfer rates. Protein binding to phospholipid vesicles containing either tritium-labeled or pyrene-labeled glucosylceramide could not be demonstrated by density gradient centrifugation or fluorescence energy transfer measurements, respectively. A specific association of the transfer protein for pyrene-labeled glucosylceramide was found when the fluorescence emission of the pyrene excimer-to-monomer ratio was measured suggesting that a portion of the fluorescent glycolipid was being sequestered from the phospholipid vesicles and was binding to the freely soluble protein.

Animals↗

PubMed1985

Organization of ganglioside GM1 in phosphatidylcholine bilayers.

Molecules of the ganglioside GM1 are randomly distributed in liquid-crystalline 1-palmitoyl-2-oleoyl phosphatidylcholine bilayers. This conclusion is based on a freeze-etch electron microscopic study using ferritin-conjugated cholera toxin and cholera toxin alone as ganglioside labels. The average number of GM1 molecules under a label is calculated by a novel method from the dependence of the fraction of bilayer area covered by the label on the mole fraction of GM1 in the bilayer.

Animals↗

PubMed1985

Spontaneous transfer of gangliotetraosylceramide between phospholipid vesicles.

The transfer kinetics of the neutral glycosphingolipid gangliotetraosylceramide (asialo-GM1) were investigated by monitoring tritiated asialo-GM1 movement from donor to acceptor vesicles. Two different methods were employed to separate donor and acceptor vesicles at desired time intervals. In one method, a negative charge was imparted to dipalmitoylphosphatidylcholine donor vesicles by including 10 mol% dipalmitoylphosphatidic acid. Donors were separated from neutral dipalmitoylphosphatidylcholine acceptor vesicles by ion-exchange chromatography. In the other method, small, unilamellar donor vesicles (20-nm diameter) and large, unilamellar acceptor vesicles (70-nm diameter) were coincubated at 45 degrees C and then separated at desired time intervals by molecular sieve chromatography. The majority of asialo-GM1 transfer to acceptor vesicles occurred as a slow first-order process with a half-time of about 24 days assuming that the relative concentration of asialo-GM1 in the phospholipid matrix was identical in each half of the donor bilayer and that no glycolipid flip-flop occurred. Asialo-GM1 net transfer was calculated relative to that of [14C]cholesteryl oleate, which served as a nontransferable marker in the donor vesicles. A nearly identical transfer half-time was obtained when the phospholipid matrix was changed from dipalmitoylphosphatidylcholine to palmitoyloleoylphosphatidylcholine. Varying the acceptor vesicle concentration did not significantly alter the asialo-GM1 transfer half-time. This result is consistent with a transfer mechanism involving diffusion of glycolipid through the aqueous phase rather than movement of glycolipid following formation of collisional complexes between donor and acceptor vesicles. When viewed within the context of other recent studies involving neutral glycosphingolipids, these findings provide additional evidence for the existence of microscopic, glycosphingolipid-enriched domains within the phospholipid bilayer.

Animals↗

PubMed1985

The effects of pressure and cholesterol on rotational motions of perylene in lipid bilayers.

Using steady-state fluorescence polarization measurements, an isothermal pressure-induced phase transition was observed in dimyristoyl-L-alpha-phosphatidylcholine multilamellar vesicles containing perylene. The temperature-to-pressure equivalence, dT/dP, estimated from the phase transition pressure, P1/2, is about 22 K X kbar-1, which is comparable to values determined from diphenylhexatriene polarization (Chong, P.L.-G. and Weber, G. (1983) Biochemistry 22, 5544-5550). In addition, we have employed a new method, introduced in this paper, to calculate the rate of in-plane rotation (Rip) and the rate of out-of-plane rotation (Rop) of perylene in lipid bilayers. The effects of pressure and cholesterol on the rotational rates of perylene in two lipid bilayer systems have been examined. They are 1-palmitoyl-2-oleoyl-L-alpha-phosphatidylcholine (POPC) multilamellar vesicles (MLV) and 50 mol% cholesterol in POPC (MLV). Rop is smaller than Rip due to the fact that the out-of-plane rotation requires a larger volume change than the in-plane rotation. Cholesterol seems not to affect Rop significantly, but pressure causes a decrease in Rop by about a factor of three. In contrast, the effects of pressure and cholesterol on Rip are less straightforward. At 1 atm cholesterol increases Rip by a factor of about two. Similarly, in the absence of cholesterol 1.5 kbar pressure essentially triples Rip. However, if both cholesterol is added and pressure is applied, Rip decreases sharply. The possible interactions between cholesterol and perylene are discussed.

Benz(a)Anthracenes↗

PubMed1985

Purification and spectroscopic properties of pyrene fatty acids.

Pyrene fatty acids are routinely purified by silica based column chromatography and analyzed on thin-layer silica plates (H.-J. Galla et al., Chem. Phys. Lipids, 23 (1979) 239-251). Although pyrene decanoic acid runs as a single spot on thin-layer chromatography (TLC), gas-liquid chromatography (GC) of the methyl ester derivatives of a representative sample revealed four separate peaks with the major component only 92% of the total. High performance reverse phase liquid chromatography (HPLC) was used to purify pyrene decanoic acid and separate the contaminants. After two passes on a C18 reverse phase HPLC column, pyrene decanoic acid is 99.98% pure by GC analysis. Absorption, fluorescence, and NMR spectra were recorded for pyrene decanoic acid and the major impurities. The results indicate that one impurity is a C10 fatty acid with an altered aromatic moiety. Two other impurities are pyrene derivatives but their acyl chains probably are not decanoic acid.

Chromatography, Gas↗

PubMed1985

Oxygen quenching of pyrene-lipid fluorescence in phosphatidylcholine vesicles. A probe for membrane organization.

Oxygen quenching has been used as an alternative method to study the temperature dependence of the apparent excimer formation constant, kdm, of N-(10-[1-pyrene]-decanoyl)-sphingomyelin (Pyr-SPM) in 1-palmitoyl-2-oleoyl-L-alpha-phosphatidylcholine (POPC) multilamellar vesicles. In conjunction with the lifetime of Pyr-SPM monomer in the absence of excimer and oxygen, kdm can be determined from the measurements of the monomer intensity as a function of oxygen concentration. The advantage of this method is that kdm can be determined without knowledge of the excimer lifetime and intensity, and without knowledge of the true concentration of oxygen in lipid bilayers. Our results show that kdm increases monotonically with temperature from 16 to 40 degrees C, becomes insensitive to temperature from 40 to 50 degrees C and increases again at 54 degrees C. The temperature-insensitive region corresponds to the temperature range of the phase transition of Pyr-SPM determined by differential scanning calorimetry. This result suggests the existence of Pyr-SPM-enriched domains in POPC vesicles. In contrast, no abrupt change in kdm with temperature occurs in the case of 1-palmitoyl-2-[10-(1-pyrenyl) decanoyl] phosphatidylcholine (Pyr-PC).

Kinetics↗

PubMed1985

Organization of glycosphingolipids in bilayers and plasma membranes of mammalian cells.

The evidence presented in this review strongly suggests that, when present as a minor component in liquid crystalline phospholipid bilayers, neutral glycosphingolipids are segregated into compositional domains of small size dispersed in the matrix phospholipid. In many instances the glycosphingolipid in the dispersed domains is in the gel state. Because these domains are in the gel state, the individual molecules escape only very slowly from the surface of the bilayer, much more slowly than do the phospholipid components. There is as yet no direct evidence that this slow escape rate is a property of neutral glycolipids in biological membrane bilayers. If it is, however, then these molecules are well suited for their putative role as cell surface markers, a role that involves them in many important biological functions. There is evidence to suggest that molecules of this type are also present in a dispersed microdomain structure on the external surface of at least some mammalian cell plasma membranes. These small domains of glycolipids with their sugar residues projecting outward from the cell surface are much like a large membrane glycoprotein when viewed from the ambient medium near the cell surface. Thus, whether the sugar residues be of glycoprotein or glycolipid origin, they are localized in groups or patches on the external surface of the cell. One important consequence of this patch structure may be in the obvious effect on the free energy of binding a ligand to a patch. Whether the ligand is mono- or polyvalent, the roughly 2 M concentration of sugar in the surface patch will cause the apparent ligand binding free energy to be substantially larger than it would be for a single isolated sugar residue on the surface. In contrast to the neutral glycosphingolipids (and sulfatides, perhaps) the available information suggests that gangliosides are not localized in small domains in model systems and most probably not in biological membranes. Capping of this type of glycosphingolipid does appear to occur under certain circumstances. However, it is almost certain that capping is not an intrinsic property of ganglioside phospholipid-bilayer systems. Although at 37 degrees C gangliosides rapidly transfer from micelles to phospholipid vesicles and to cell membranes, nothing is known about the rates at which this class of molecules leave a phospholipid bilayer. Their known biological functions on the cell surface appear to require that they leave very slowly, if at all, as do the neutral glycosphingolipids. The glycosphingolipids are, by virtue of their polysaccharide moeity, a unique class of lipids and cell surface components.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1984

Glycolipid transfer protein from bovine brain.

Glycolipid transfer protein from bovine brain has been purified partially by ammonium sulfate precipitation, CM-52 ion-exchange, and Sephadex G-75 column chromatography. Both pyrene-labeled and tritium-labeled glucocerebrosides have been used to study the kinetics of protein-mediated transfer between donor and acceptor vesicles. Protein accelerates glucocerebroside transfer but does not accelerate phospholipid transfer. In colyophilized small sonicated vesicles (10% glucocerebroside, 90% 1-palmitoyl-2-oleoyl-phosphatidylcholine) about two-thirds of the glycolipid is transferred in 2 h and the remaining one-third does not transfer (up to 5 h). For donor and acceptor vesicles made of dipalmitoylphosphatidylcholine or 1-palmitoyl-2-oleoyl-phosphatidylcholine, glucocerebroside (10% in donors) is transferred rapidly only when both the donor and acceptor matrix phospholipids are in the liquid-crystalline state. If either donor or acceptor vesicles are in the gel state, transfer protein mediated transfer is much reduced. The amount of transfer protein bound specifically to glucocerebroside-containing vesicles is nearly equal above and below the matrix phospholipid phase transition temperature. Bound protein transfers glucocerebroside upon addition of acceptor vesicles.

Animals↗

PubMed1983

Kinetics of transfer of gangliosides from their micelles to dipalmitoylphosphatidylcholine vesicles.

Two aspects of the kinetics of transfer of ganglioside from micelles to dipalmitoylphosphatidylcholine vesicles have been examined: (i) The first aspect is the rate of transfer of ganglioside from micelles at very low ganglioside/phospholipid ratios. Under these conditions the rate of incorporation into vesicles is independent of the vesicle concentration, indicating that transfer occurs by diffusion of ganglioside molecules through the aqueous phase and not by collision of micelles and vesicles. The initial transfer of monosialoganglioside is slower (t 1/2 = 2 h) than that of trisialoganglioside (t 1/2 = 0.5 h). The rate of transfer decreases during the transfer process. This decrease in rate depends on the character of the micelles and not on the ganglioside content of acceptor vesicles. The initial rate of transfer decreases sharply with decreasing temperature. (ii) The second aspect is the rate of transfer of ganglioside from micelles to phospholipid vesicles at high ganglioside/phospholipid ratios. In the presence of excess ganglioside, the level of incorporation into vesicles saturates when the ganglioside content of the vesicles reaches 12-15 mol %. This saturation level is not markedly dependent on the number of sialic acid residues in the ganglioside.

Colloids↗

PubMed1982

Organization of the glycosphingolipid asialo-GM1 in phosphatidylcholine bilayers.

An affinity purified monovalent ferritin conjugate of Ricinus communis agglutinin (RCA 60) is used with freeze-etch electron microscopy to study the ultrastructural localization of the glycosphingolipid asialo-GM1 in multilamellar phosphatidylcholine liposomes. Dimyristoylphosphatidylcholine (DMPC) liposomes containing up to 20 mol% asialo-GM1 and quenched below the main transition temperature show a striking linear localization of ferritin-RCA 60 between phospholipid ridges. The glycosphingolipid localization is similar to that postulated for up to 20 mol% cholesterol in pure phosphatidylcholine bilayers by Copeland, B.R. and McConnell, H.M. (Biochim. Biophys. Acta, 599, 95-109 (1980)). Above the main phase transition temperature, asialo-GM1 appears to be organized into clusters, especially in palmitoyloleoylphosphatidylcholine (POPC) liposomes. This clustered distribution of glycosphingolipids seen above the phase transition temperature suggests that this type of lipid may exhibit compositional domain structure in biological membranes.

Chemical Phenomena↗

PubMed1982

Fusion of dipalmitoylphosphatidylcholine vesicles at 4 degrees C.

Small sonicated dipalmitoylphosphatidylcholine vesicles when incubated at 4 degrees C and high concentrations are shown to fuse completely to vesicles about 700-A diameter in 7 days, and these further fuse to about 950 A diameter vesicles after 3-4 weeks. The 950 A diameter vesicles are spherical, homogeneous, mostly unilamellar, have an internal aqueous space about 10 times that of small vesicles, and are stable for at least 6 months. The 950-A vesicles are characterized by agarose gel chromatography, freeze-fracturing electron microscopy, trapped volume measurements, differential scanning calorimetry, and diphenylhexatriene fluorescence polarization.

Chemical Phenomena↗

PubMed1982

Aggregation of dipalmitoylphosphatidylcholine vesicles.

Quasi-elastic and 90 degrees light scattering were used to study the aggregation of dipalmitoylphosphatidylcholine vesicles at temperatures below the gel-liquid-crystalline phase transition as a function of concentration, temperature, and size. Increased vesicle concentration did not appreciably change aggregate size but did change the total number of aggregates in a manner consistent with a bimolecular collisional mechanism for the conversion of aggregates to fused vesicles. Increased temperature decreased aggregation, indicating that the disaggregation rate constant increased faster than the aggregation rate constant. As a function of size, aggregation decreased slightly from small to 700 A diameter vesicles and increased considerably for 950 A diameter vesicles. A model of the interaction of small vesicles below the gel-liquid-crystalline phase transition is presented in which aggregation precedes fusion and collision between aggregates triggers fusion.

Chemical Phenomena↗

PubMed1982

Effect of cytochrome b5 on the transbilayer distribution of phospholipids in model membranes.

The transbilayer distribution of phosphatidylethanolamine was assessed in phosphatidylcholine-phosphatidylethanolamine vesicles that contained various amounts of cytochrome b5. The small vesicles, made by sonication, and the large vesicles, made by ethanol injection, were fractionated by centrifugation before cytochrome b5 was asymmetrically incorporated into the bilayer. The mole ratio of phospholipid to protein ranged from 280 to 560 in the small vesicles and from 100 to 500 in the large vesicles. The phosphatidylethanolamine distribution, determined by chemical labeling with trinitrobenzenesulfonic acid, was assessed in vesicles the contained intact cytochrome b5 molecules and in vesicles where only the hydrophobic tail remained associated with the bilayer. At every phospholipid to protein ratio examined, the transbilayer distribution of phosphatidylethanolamine in either the small or large unilamellar vesicles was not significantly different from the distribution in control vesicles that contained no protein. Ethanol was added to some cytochrome b5-vesicle preparations (20% v/v) in an attempt to facilitate rearrangement of the phospholipids. No differences in the transbilayer distribution were observed. These results are discussed in terms of transbilayer equilibrium and the perturbation induced by the protein.

Cytochromes↗

PubMed1982

Glucocerebroside transfer between phosphatidylcholine bilayers.

We have studied the kinetics of transfer of glucocerebroside between phospholipid bilayers by using pyrene and 3H-labeled glucocerebroside incorporated into dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC) bilayers. Pyrene-labeled glucocerebroside (PyrCer) molecules are able to form an excited complex (eximer, E) between a PyrCer in the ground state and an excited monomer (M). When vesicles contained a known amount of PyrCer (donors) are incubated with unlabeled vesicles (acceptors), transfer of PyrCer from donor to acceptor populations is reflected in a decrease of the observed E/M intensity ratio. The results obtained from these studies show that the half-time of transfer from donor DMPC-PyrCer vesicles to acceptor DMPC vesicles is greater than 30 days at 37 degrees C. This very slow transfer of glucocerebroside was confirmed by using tritiated glucocerebroside incorporated into small unilamellar DPPC donor vesicles incubated with large unilamellar DPPC acceptor vesicles above the phase transition. Separation of the two vesicle populations by molecular sieve chromatography at 45 degrees C shows a half-time for transfer of approximately 32 days. We conclude that, in contrast to the results obtained for phosphatidylcholines [Roseman, M., & Thompson, T. E. (1980) Biochemistry 19, 439], glucocerebroside does not rapidly transfer between bilayers under these conditions.

Cerebrosides↗

PubMed1982

Effect of phospholipid oxidation products on transbilayer movement of phospholipids in single lamellar vesicles.

Single lamellar phosphatidyl[methyl-2H]choline vesicles were incubated with an excess of unlabeled phosphatidylcholine vesicles or phosphatidylcholine-cholesterol vesicles containing 8 mol % glucuronosyldiglyceride. Incubation of the two vesicle populations was performed in the presence or absence of a purified phosphatidylcholine exchange protein. The negatively charged glycolipid donor vesicles could be completely removed by column chromatography on DEAE-Sephacel. Following incubation with exchange protein and subsequent fractionation, the -N(CD3)3 phosphatidylcholine acceptor vesicles exhibited a 61-73% enrichment of the unlabeled phosphatidylcholine in the outer monolayer. Upon incubation in an air atmosphere, no appreciable transbilayer movement of the outer monolayer -N(CH3)3 phosphatidylcholine was observed for at least 5 days. Between days 5 and 7, however, extensive transbilayer movement occurred, leading to an outer monolayer/inner monolayer phosphatidylcholine ratio of 2.1 on day 7. In phosphatidylcholine-6 mol % cholesterol vesicles treated similarly, the outside/inside ratio of the unlabeled phospholipid was 6.7, suggesting a much smaller percentage of transbilayer movement. The loss of transbilayer asymmetry which occurred during a 36-h period after day 5 could be estimated at the upper limit, t 1/2 approximately 7.3 h for phosphatidylcholine vesicles and t 1/2 approximately 53 h for phosphatidylcholine-cholesterol vesicles. The actual rates for transbilayer movement, however, were likely more rapid. Transbilayer movement occurred at a time period when oxidized phospholipid breakdown products had reached critical levels.

Androgen-Binding Protein↗

PubMed1982

Transition of a liquid crystalline phosphatidylcholine bilayer to the gel phase in a vesicle reduces the internal aqueous volume.

The liquid crystalline to gel phase transition in phospholipid bilayers is associated with a marked reduction in the area per phospholipid molecule. Geometric considerations based on published data suggest that this decrease in molecular area is accompanied by a reduction in the internal aqueous volume trapped within a unilamellar bilayer vesicle. This volume reduction, which depends upon the shape of the vesicle, is shown to be between 23 and 60 percent. We have observed a 25 to 30 percent reduction in the internal aqueous volume of unilamellar vesicles about 700 A in diameter formed from dipalmitoylphosphatidylcholine using the self-quenching of 6-carboxyfluorescein trapped within this compartment.

Fluoresceins↗