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R M Epand

Publications and source records attributed to R M Epand.

At least 109 records · Page 6Linked to original sources
PubMed1995

Mechanisms for the modulation of membrane bilayer properties by amphipathic helical peptides.

The amphipathic helix, in which hydrophobic and hydrophilic residues are grouped on opposing faces, is a structural motif found in many peptides and proteins that bind to membranes. One of the physical properties of membranes that can be altered by the binding of amphipathic helices is membrane monolayer curvature strain. Class A amphipathic helices, which are present in exchangeable plasma lipoproteins, can stabilize membranes by reducing negative monolayer curvature strain; proline-punctuated class A amphipathic helical segments are particularly effective in this regard. This property is suggested to be associated with some of the beneficial biological effects of this protein. On the other hand, lytic amphipathic helical peptides can act by increasing negative curvature strain or by forming pores composed of helical clusters. Thus, different amphipathic helical peptides can be membrane stabilizing or be lytic to membranes, depending on the structural motif of the helix, which in turn determines the nature of its association with membranes. Features of these peptides that are responsible for their specific properties are discussed.

Amino Acid Sequence↗
PubMed1995

Mechanism of liposome destabilization by polycationic amino acids.

Polycationic amino acids induce the leakage and fusion of liposomes containing anionic lipids. We have investigated the nature and extent of the changes in membrane physical properties caused by these polypeptides which could result in the observed membrane destabilization. We found that in the range of pH 5 to pH 7 both poly-L-histidine and poly-L-lysine were ineffective in shifting the bilayer to hexagonal phase transition temperature of dielaidoylphosphatidylethanolamine, either in the presence of absence of 1-palmitoyl-2-oleoylphosphatidylserine. We also studied the gel to liquid crystalline phase transition properties of 1:1 mixtures of phosphatidylserine and phosphatidylethanolamine, both in dimyristoyl forms as well as the 1-palmitoyl-2-oleoyl forms, as a function of pH and in the presence and absence of polycationic amino acids. We observed that these two lipids were largely miscible at all pH values and in the presence and absence of the polypeptides. However, there was some increased tendency for phase separation at higher pH and in the absence of polypeptide. Thus neither changes in curvature strain nor lateral phase separation induced by the polycationic amino acids could account for their marked ability to induce leakage and fusion. Phosphatidylethanolamine labelled with pyrene on one of the acyl chains gives rise to fluorescent emission from both monomer and excimer forms. The ratio of emission intensity from these two forms is indicative of lateral phase separation and the degree of lateral mobility of this probe. In equimolar mixtures of the 1-palmitoyl-2-oleoyl forms of phosphatidylserine and phosphatidylethanolamine in the liquid crystalline phase at 30 degrees C we find little effect of pH on the ratio of excimer to monomer emission intensity. However poly-L-lysine markedly lowers the fraction of excimer emission from these liposomes through the pH range from 5 to 7. Poly-L-histidine lowers the excimer to monomer emission ratio at pH 5 but not at pH 7. This is opposite to what one would expect for lateral phase separation and is interpreted at being the consequence of the polypeptide lowering the rate of lateral diffusion of the lipids. This effect of poly-L-histidine is observed over a range of temperatures from 0 to 40 degrees C in both gel and liquid crystalline phases. There is no evidence from the behaviour of the pyrene fluorescent probe for lipid interdigitation.(ABSTRACT TRUNCATED AT 400 WORDS)

Calorimetry, Differential Scanning↗
PubMed1995

Estimation of cholesterol sulphate in blood plasma and in erythrocyte membranes from individuals with Down's syndrome or diabetes mellitus type I.

OBJECTIVES: Plasma and erythrocyte membrane cholesterol sulphate (CS) were measured in patients suffering from diabetes and Down's syndrome. DESIGN AND METHODS: The procedure for separation and determination of CS comprised HPTLC (high-performance thin-layer chromatography) and densitometry. RESULTS: The mean plasma and RBC membranes CS concentrations (+/- SD) of the control group (n = 16) was 188 +/- 47 micrograms/dL and 343 +/- 57 micrograms/10(12) RBC, respectively. In 15 patients with diabetes and 12 Down's syndrome patients substantially higher CS levels were found (diabetes: plasma-348 +/- 60 micrograms/dL; RBC membranes-646 +/- 113 micrograms/10(12) RBC; Down's syndrome: plasma-245 +/- 54 micrograms/dL; RBC membranes 427 +/- 74 micrograms/10(12) RBC). Analysis of variance and multiple comparison (Newman-Keuls test) show statistically significant differences between all samples both for erythrocytes, F(2.41) = 52.24, p < 0.05, and plasma, F(2.41) = 34.92, p < 0.05. CONCLUSIONS: It is postulated that differences in CS levels may contribute to changes of erythrocyte properties in these pathological states.

Adult↗
PubMed1995

Lipid-mediated a-factor interactions with artificial membranes.

Our understanding of the cellular export of a-factor and its interaction with the receptor do not yet allow for a description of the phenomena on a molecular level. Synthesis of a-factor analogs and biophysical studies of the lipopeptides in the presence of artificial membranes provide insights which can be analyzed with respect to the biological potency of the molecules. It is through the study of the interaction of the lipopeptides with membranes at varying levels of complexity that we will be able to develop a molecular description of the biological processes.

Alkyl and Aryl Transferases↗
PubMed1995

Divergent structural requirements exist for calcitonin receptor binding specificity and adenylate cyclase activation.

The basis of the high potency of salmon calcitonin (sCT) in radioligand binding competition and cAMP accumulation studies with cloned calcitonin (CT) receptors from rats, pigs, and humans was examined using two sets of CT analogues, i.e., chimeric sCT/human CT (hCT) analogues and analogues of sCT with differing capacities to form an amphipathic alpha-helix. In competition for 125I-sCT binding the following relative specificities were observed for the chimeric peptides: rat C1a CT receptor, sCT > or = (1-16)hCT/(17-32)sCT (ACT-15) > (1-16)sCT/(17-32)hCT (ACT-27); rat C1b CT receptor, sCT >> ACT-15 > ACT-27; hCT receptor, sCT = ACT-15 > ACT-27; porcine CT receptor, sCT > ACT-27 > ACT-15. In contrast, in ligand-induced cAMP accumulation studies the relative efficacies were as follows: rat C1a CT receptor, sCT = ACT-15 > ACT-27; rat C1b CT receptor, sCT = ACT-15 > ACT-27; hCT receptor, sCT = ACT-15 > or = to ACT-27; porcine CT receptor, sCT = ACT-15 = ACT-27. The data demonstrate that residues present in the carboxyl-terminal half of sCT are more important for binding competition with the rat C1a, rat C1b, and human CT receptors, whereas residues in the amino-terminal half of sCT are more important for binding competition with the porcine CT receptor. Carboxyl-terminal sCT residues are also important for full potency in adenylate cyclase activation with the rat C1a and rat C1b CT receptors but are less important for activation via the hCT receptor. The disparity in the relative potencies of the peptides in studies of binding competition and cAMP accumulation is suggestive of significant differences in the relative affinities of the peptides for active and inactive conformations of the CT receptor. The use of sCT analogues with varying capacities to form alpha-helices also revealed divergence in the responses of different receptors. This was most apparent for the stimulation of cAMP production by the rat receptor isoforms C1a and C1b. In cells expressing the C1a receptor, the helical analogues sCT and des-Ser2-sCT were equipotent with [Gly8]-des-Leu19-sCT and des-1-amino-[Ala1,7,Gly8]-des-Leu19 sCT, analogues that have reduced or absent helical structure, respectively. In contrast, the nonhelical analogues were 100-1000-fold less potent than sCT and des-Ser2-sCT at the C1b receptor. In general, reduction in the ability of sCT analogues to form helix structures had a greater impact on the potency of the analogues in competition for 125I-sCT binding than in cAMP accumulation.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenylyl Cyclases↗
PubMed1994

Membrane orientation of the SIV fusion peptide determines its effect on bilayer stability and ability to promote membrane fusion.

The amino terminal segment of the gp32 glycoprotein of SIV has been identified as an important region for membrane fusion. A synthetic dodecapeptide corresponding to this amino terminal segment, SIVwt, can promote the fusion of liposomes. This peptide inserts at an oblique angle into the membrane. If the amino acid sequence of this peptide is changed, while maintaining the same amino acid composition, the resulting peptide, SIVmutV, no longer promotes fusion and it is oriented perpendicular to the plane of the bilayer. In the present work we demonstrate that SIVwt, but not SIVmutV, can lower the bilayer to hexagonal phase transition temperature of model membranes composed of dipalmitoleoyl phosphatidylethanolamine. In addition the SIVwt promotes the formation of structures which give rise to isotropic 31P NMR signals in mixtures with monomethyldioleoyl phosphatidylethanolamine. These structures are not formed with SIVmutV and their formation with SIVwt is suppressed with lysophosphatidylcholine. Taken together these results suggest that the observed correlation between oblique insertion of viral fusion peptides into membranes and their fusogenicity may be a consequence on these peptides increasing negative monolayer curvature.

Amino Acid Sequence↗
PubMed1994

Relationship between the infectivity of influenza virus and the ability of its fusion peptide to perturb bilayers.

The amino terminal segment of the HA2 protein of influenza virus has been identified as an important region for membrane fusion. We demonstrate that there is an association between the ability of model peptides corresponding to this region of the HA2 protein to promote the formation of inverted phases and the fusogenicity of the intact virus. The wild type virus can fuse to membranes at pH 5 but not at pH 7.4. We show that the fusion peptide of the wild type virus lowers the bilayer to hexagonal phase transition temperature of dipalmitoleoylphosphatidylethanolamine at pH 5 but at pH 7.4 it raises this transition temperature. In addition it has been shown that site specific mutagenesis resulting in the substitution of Gly with Glu leads to a loss of viral infectivity. These same amino acid substitutions in the viral fusion peptide result in the peptide no longer being able to lower the bilayer to hexagonal phase transition temperature, even at acidic pH. The wild type fusion peptide promotes the formation of structures which give rise to isotropic 31P NMR spectra at pH 5.0 but not at pH 7.4. The two altered sequences of the fusion peptide, corresponding to the Glu to Gly mutations, do not promote isotropic 31P NMR signals at acidic pH. These results indicate that the fusogenicity of influenza virus is dependent, in part, on the ability of the amino terminal region of HA2 to disrupt stable bilayer packing and to induce curvature strain corresponding to inverted phase structures.

Amino Acid Sequence↗
PubMed1994

Alamethicin pyromellitate: an ion-activated channel-forming peptide.

The synthesis and characterization of alamethicin pyromellitate (Alm-PM), a derivative of the channel-forming peptide alamethicin bearing three negative charges at the C-terminus, is described. The self-association of Alm-PM in small unilamellar vesicles of dioleoylphosphatidylcholine (DOPC), monitored using circular dichroism (CD) spectroscopy, occurs much less readily than the self-association of unmodified alamethicin. Channel formation by Alm-PM also occurs less readily and exhibits a higher voltage threshold for activation in planar lipid bilayers and in lipid vesicles. An increase in the salt concentration, and particularly the addition of calcium ions, promotes Alm-PM self-association as monitored by CD spectroscopy. Calcium also facilitates channel formation by Alm-PM both in planar lipid bilayers and in lipid vesicles by lowering the voltage threshold for activation. Thus Alm-PM behaves as an ion-activated ion channel. These results indicate that the self-association of alamethicin-like peptides in membranes is critical for channel formation and that transmembrane flip-flop of peptide helices is not required. In addition, these results demonstrate that the activity of channel-forming peptides may be controlled by controlling the process of self-association.

Alamethicin↗
PubMed1994

Cationic liposomes for direct gene transfer in therapy of cancer and other diseases.

Cationic liposomes can mediate efficient delivery of DNA and DNA/protein complex to mammalian cells in vitro and in vivo. Cationic cholesterol derivatives mixed with phosphatidylethanolamine and sonicated to form small unilamellar vesicles can complex with DNA and mediate the entry into the cytosol from the endosome compartment. One of the liposome formulations, DC-Chol liposomes, is used in a gene therapy clinical trial for melanoma. Recently, we exploited these cationic liposomes for the delivery of trans-activating protein factors to regulate and control the expression of delivered transgenes in a protein dose-dependent manner. Bacteriophage T7 RNA polymerase was co-delivered with a reporter gene under the control of T7 promoter to allow cytoplasmic expression of the gene. Human immunodeficiency virus-1 transactivating protein was also codelivered with a reporter gene under the control of HIV-1 long terminal repeat. Finally, human tumor cells selected for cis-platin resistance or isolated from patients who have failed cis-platin therapy are highly transfectable with cationic liposomes. These results suggest a serial therapy protocol with cis-platin and gene therapy for malignancy.

Animals↗
PubMed1994

Characterization of the calcium-binding site that regulates association of protein kinase C with phospholipid bilayers.

Association of calcium-dependent isotypes of protein kinase C (PKC) with a phospholipid bilayer is regulated by a single Ca(2+)-binding site. The dependence of PKC association with phosphatidylserine-containing membranes on the concentration of Ca2+ is linear in the submicro- to submillimolar range. The Ca(2+)-regulated association of PKC with the membrane is sensitive to the factors that alter the diffuse double-layer potential produced by anionic lipids such as phosphatidylserine (PS). This indicates that the Ca(2+)-binding site on the membrane-bound enzyme senses a higher concentration of Ca2+ than is present in bulk solution. This is a consequence of the accumulation of Ca2+ in the layer adjacent to the plane of the membrane by the double-layer potential. Calculations based on the Gouy-Chapman-Stern theory of the diffuse double layer yielded a unique value of the Ca2+ dissociation constant for the Ca(2+)-PKC-bilayer complex equal to approximately 700 nM. The soluble form of the enzyme has a 3.5 order of magnitude lower affinity for Ca2+. The free energy of interaction between the Ca(2+)- and PS-binding sites is large (approximately 5 kcal/mol). In contrast, the interaction between the diacylglycerol-binding site and either the Ca(2+)- or PS-binding site appears to be weak.

Amino Acid Sequence↗
PubMed1994

The effects of membrane physical properties on the fusion of Sendai virus with human erythrocyte ghosts and liposomes. Analysis of kinetics and extent of fusion.

A number of amphiphiles which raise the bilayer to hexagonal phase transition temperature (TH) of phosphatidylethanolamine (PE) have been shown to inhibit viral fusion. In this study we have further evaluated the mechanism of this inhibition. Several anionic amphiphiles, including cholesterol sulfate, a component of mammalian plasma membranes, lower the final extent of Sendai virus fusion with both human erythrocyte ghosts and liposomes composed of PE and 5% of the ganglioside, GD1a. A cationic amphiphile slightly increased the final extent of fusion. The fusion rate constant is not greatly affected by the presence of as much as 20% cholesterol sulfate or other charged amphiphiles. The zwitterionic amphiphile, cholesterol phosphorylcholine has no effect on the final extent of fusion but it lowers the fusion rate constant. This amphiphile is potent in raising TH. The amphiphile cholesterol hemisuccinate (CHEMS) stabilizes the bilayer relative to the hexagonal phase at neutral pH, while at acidic pH the formation of the hexagonal phase is promoted. When CHEMS is added to vesicles of egg PE containing 5% GD1a, the rate of Sendai virus fusion is little affected at neutral pH but the rate is significantly enhanced at pH 5.0. These results demonstrate that viral fusion can be modulated, in part, by the tendency of the membrane to convert to the hexagonal phase.

Animals↗
PubMed1994

Analogues of tetramethylpyrazine affect membrane fluidity of liposomes: relationship to their biological activities.

Several structurally similar pyrazine derivatives, tetramethylpyrazine, triethylpyrazine and tetraethylpyrazine have previously been found to inhibit a broad spectrum of plasmalemma-associated biological activities of various tissues, including ion channels and membrane receptors in a given order of potency that increases with increasing bulkiness and hydrophobicity of these drugs. Our earlier speculation that the multiple membrane effects elicited by these pyrazine derivatives were associated with changes in the bulk physical properties of the biological membranes in the presence of these drugs was confirmed by fluorescence polarization and electron spin resonance studies. Since biological membranes contain a complicated mixture of a large variety of lipids and proteins, we chose to study the molecular properties of the interaction of these three pyrazine derivatives using a simple model system composed of synthetic phospholipids. We measured the effects of the drugs on the fluidity (or microviscosity) of these model membranes, as well as determining the phase-transition properties of the model membranes with the use of fluorescence polarization, electron spin resonance and differential scanning calorimetry. All the methods employed yielded qualitatively similar results. Tetramethylpyrazine, the smallest and the least hydrophobic amongst these three drugs, had minimal or no effect on the phase-transition of phospholipids based on temperature dependence studies, whereas tetraethylpyrazine, the largest and the most hydrophobic of the three, elicited the most potent effect in a concentration-dependent manner. These results could be attributed to the amount of incorporation of these pyrazine derivative into the liposomes, an interaction which occurs more favourably at temperatures above the gel to liquid crystalline phase-transition temperature.(ABSTRACT TRUNCATED AT 250 WORDS)

Calorimetry, Differential Scanning↗
PubMed1994

Fusion of influenza to liposomes is not inhibited by aliphatic primary alcohols.

Reports of the antiviral activity of aliphatic alcohols led us to investigate the effects of aliphatic alcohols, from 10 to 20 carbons in length, on the phase transition behaviour of model phospholipids and on the fusion of influenza to liposomes. Contrary to the effects of many other antiviral agents, we find that alcohols are potent promoters of the inverted hexagonal phase. However, we also find that aliphatic alcohols have little effect on influenza fusion to liposomes. Eicosanol is the only aliphatic alcohol tested which substantially increases in fusion of influenza virus. We also find that long chain alcohols display multi-component bilayer to hexagonal phase transitions at higher mole fractions. This suggests that eicosanol may be facilitating fusion by creating defects between alcohol-rich and alcohol-poor regions of the lipid bilayer.

Alcohols↗
PubMed1994

Calorimetric detection of curvature strain in phospholipid bilayers.

Phospholipids in biological membranes are arranged as bilayers. When constrained to pack into planar bilayers, certain phospholipids will form unstable structures as a consequence of their molecular shape and noncovalent bonding. This produces curvature strain which may provide energy for certain membrane processes. We demonstrate that an exothermic process associated with the relief of curvature strain can be detected calorimetrically. The enthalpy for the incorporation of a few percent lysophosphatidylcholine into large unilamellar vesicles of monomethyldioleoylphosphatidylethanolamine at pH 7.4 is exothermic but it is endothermic for stable bilayers such as this same lipid at pH 9 or dioleoylphosphatidylcholine at pH 7.4 or 9. The addition of lysophosphatidylcholine to monomethyldioleoylphosphatidylethanolamine at pH 7.4 is exothermic only for the addition of the first few percent of lysophosphatidylcholine and then it becomes endothermic. The size of the exothermic heat change is sensitive to changes in temperature, while the endothermic processes are relatively temperature-insensitive. The exothermic heat is also larger when 1 or 2 mol % of diolein is incorporated into vesicles of monomethyldioleoylphosphatidylethanolamine. These results are all consistent with the exothermic process corresponding to the relief of curvature strain in bilayers having a tendency to convert to the hexagonal phase. It provides a demonstration that considerable energy may be released upon the incorporation of certain molecules into membranes which have a low radius of spontaneous curvature.

Biophysical Phenomena↗
PubMed1994

HDL and apolipoprotein A-I protect erythrocytes against the generation of procoagulant activity.

The appearance of anionic lipids on the extracellular surface of cells is required for the formation of the procoagulant complex that leads to the activation of prothrombin. Procoagulant activity would be expected to be inhibited by substances that stabilize the membrane structure and hence inhibit the transbilayer diffusion of phosphatidylserine from the cytoplasmic to the extracellular surface of the plasma membrane. The generation of procoagulant activity in human erythrocytes by A23187 and Ca2+ is inhibited by apolipoprotein A-I, its amphipathic peptide analogues, and high-density lipoprotein (HDL). These agents do not inhibit the Ca2+ loading of erythrocytes by A23187, nor do they inhibit the activation of prothrombin once the cells have been incubated at 37 degrees C with A23187 and Ca2+. Transbilayer diffusion of fluorescently labeled phosphatidylserine is inhibited by apolipoprotein A-I. These findings indicate that class A amphipathic helixes as well as lipoprotein particles and liposomes inhibit the transbilayer diffusion of phospholipids and procoagulant activity. This activity may contribute to the protective role of HDL against arteriosclerosis and thrombosis.

Amino Acid Sequence↗
PubMed1994

Mixing rates can markedly affect the kinetics of peptide-induced leakage from liposomes.

Assays on vesicle aqueous content leakage are widely used in the study of peptide-lipid interactions. We found this assay to be affected by the mode of mixing vesicle and peptide solutions. This effect can lead to artifactual conclusions regarding the lytic activity of peptides. We demonstrate that the source of this artifact is that fast (millisecond range) peptide-membrane association creates a nonhomogeneous distribution which exists sufficiently long after peptide addition to markedly alter the course of leakage. Mixing problems can be overcome by using a stopped flow apparatus. It can also be diminished by switching to injecting a small volume of vesicle suspension into a large volume of peptide solution. Mixing rates are rarely considered in literature reports of peptide effects on liposomes. The same artifacts can also take place in a number of other assays of the activity of membrane active peptides on liposomes and thus their consideration is of general importance.

Artifacts↗
PubMed1993

Lysophosphatidylcholine mediates the mode of insertion of the NH2-terminal SIV fusion peptide into the lipid bilayer.

We report here on the interaction of a synthetic 12 residue peptide corresponding to the N-terminal sequence of gp32 from SIV with phospholipid bilayers. This peptide has been shown to induce lipid mixing of PC/PE/SM/Chol LUV (large unilamellar vesicles) at pH 7.4 and 37 degrees C [(1992) in: Advances in Membrane Fluidity, vol. 6, pp. 365-376, Wiley-Liss]. In the present study, this fusion process was inhibited by the addition of lysophosphatidylcholine (lysoPC) to the lipid bilayer of PC/PE/SM/Chol LUV. Fourier transform infrared spectroscopy (FTIR) reveals that the orientation of the SIV fusion peptide with respect to the lipid acyl chains depends on the presence of lysoPC in the lipid bilayer but that the peptide secondary structure and the amount of lipid-associated peptides do not depend on the lipid composition. The peptide is obliquely inserted into the lipid bilayer of vesicles without lysoPC, whereas it is oriented parallel to the lipid-water interface in the vesicles containing lysoPC. The data provide evidence that the orientation of the SIV fusion peptide depends on the lipid composition, and that this mediates its fusogenic activity.

Kinetics↗