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Biomedical subjects

R M Epand

Publications and source records attributed to R M Epand.

At least 127 records · Page 7Linked to original sources
PubMed1993

Reciprocal effects of apolipoprotein and lytic peptide analogs on membranes. Cross-sectional molecular shapes of amphipathic alpha helixes control membrane stability.

Apolipoprotein (class A) amphipathic helixes are postulated to act as detergents by virtue of their cross-section being wedge-shaped. Using computer analysis of naturally occurring class A and lytic (class L) amphipathic helixes, we designed two archetypical model peptides. Analogs of these two peptides, incorporating substitutions or modifications of interfacial or basic residues, had the following effects. Class A peptides stabilized bilayer structure, reduced leakage from large unilamellar vesicles and erythrocytes, and inhibited lysis induced by class L peptides. Class L peptides destabilized bilayer structure in model membranes and increased binding of class A peptides to erythrocytes. The ability of class L analogs to lyse membranes and induce inverted lipid phases was reduced by either decreasing the bulk of an interfacial residue, increasing the angle subtended by the polar face, or increasing the bulk of the basic residues. The ability of the class A analog to stabilize bilayer structure and inhibit erythrocyte lysis by class L peptides was enhanced by methylating the Lys residues. These results can be explained by a model that we term the reciprocal wedge hypothesis. By analogy to the reciprocal effects of phospholipid shapes on membrane structure, we propose that the wedge shape of class A helixes stabilizes membrane bilayers, whereas the inverted wedge shape of class L helixes destabilizes membrane bilayers, and, thus, one class will neutralize the effect of the other class on membranes.

Amino Acid Sequence↗
PubMed1993

Direct evidence for the partial dehydration of phosphatidylethanolamine bilayers on approaching the hexagonal phase.

X-ray diffraction studies on oriented multilayers of 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE) in the lamellar gel (L beta) and inverted hexagonal (HII) phases at various temperatures (5-50 degrees C) and relative humidities (0-100%) are reported. One-dimensional electron density profiles of the L beta phase bilayers were constructed to a resolution of better than 4 A using direct methods to solve for the phase problem. In addition, the electron density profiles were fitted favorably using a model in which the atomic groups were assumed to be Gaussian distributed [Wiener, M. C., & White, S. H. (1992) Biophys. J. 61, 434-447]. The X-ray data clearly demonstrate that, at 100% relative humidity (RH), POPE samples exist in two distinct L beta phases, differing primarily in the amount of water between the lamellae. As the hexagonal phase transition temperature is approached, 100% RH POPE samples partially dehydrate, releasing approximately 5 water molecules per phospholipid and experiencing on average a 3-A decrease in repeat spacing. The lower temperature hydrated L beta phase POPE electron density distribution resembles that obtained from the L beta phase 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) bilayers and is unlike the partially dehydrated POPE bilayers.

Desiccation↗
PubMed1993

Structural requirements for the inhibition of membrane fusion by carbobenzoxy-D-Phe-Phe-Gly.

The peptide ZfFG is known to inhibit non-bilayer phase formation as well as vesicle-vesicle and viral fusion. In order to ascertain some of the properties or structural features of this peptide which were important for the inhibition of membrane fusion, the blocking group was transferred from the amino to the carboxyl end to make fFGOBz. The fFGOBz lowered the bilayer to hexagonal phase transition temperature of dielaidoylphosphatidylethanolamine and it promoted the formation of isotropic phases in monomethyldioleoylphosphatidylethanolamine. The promotion of non-bilayer phases by fFGOBz appeared to be enhanced by a charged terminal amino group as higher pH or formylation of the amino group both decreased the effectiveness of this peptide to induce formation of the hexagonal phase. With the monomethyldioleoylphosphatidylethanolamine, the fFGOBz also promoted vesicle leakage and fusion as measured by lipid intermixing. The fFGOBz did not inhibit the formation of lipid structures of high curvature, resulting from sonication of phosphatidylcholine, as did ZfFG. Thus, the effects of fFGOBz on membranes are in sharp contrast to those of ZfFG and more closely resemble the behaviour of larger fusion peptides corresponding to the amino-terminal segment of viral fusion proteins. Our results demonstrate that having the carbobenzoxy group on the amino-terminus of fFG is important for giving the peptide derivative the property of inhibiting membrane fusion.

Amino Acid Sequence↗
PubMed1993

Role of prenylation in the interaction of the a-factor mating pheromone with phospholipid bilayers.

We have studied the interaction between phospholipids and a-factor (YIIKGVFWDPAC-[Farn]OMe), S-alkylated forms of a-factor with the farnesyl group substituted by methyl, hexadecanyl, or benzyl groups, and truncated forms of this lipopeptide. Circular dichroism studies suggest that, despite its lack of farnesylation, S-methyl-a-factor is incorporated into vesicles of dimyristoylphosphatidylcholine in a conformation similar to that which a-factor adopts in this membrane. However, studies of the intrinsic fluorescence of the Trp residues of these peptides indicate that this residue is more deeply imbedded into the bilayer in the case of the farnesylated peptide. The a-factor is more effective in raising the bilayer to the hexagonal phase transition temperature of dielaidoylphosphatidylethanolamine than is the S-methyl-a-factor. This bilayer-stabilizing ability is also reflected in a-factor inhibiting leakage from vesicles of N-methyldioleoylphosphatidylethanolamine. Studies on a-factor analogs permit the conclusion that the bilayer-stabilizing effect of a-factor is not solely a consequence of its greater partitioning into the membrane but is also a consequence of the degree of penetration into the bilayer and the specific conformation of the peptide at the membrane interface. These results indicate that the farnesyl group alone, in the absence of cellular factors, bestows a particular physical interaction with membranes.

Amino Acid Sequence↗
PubMed1993

Increased accumulation of drugs in a multidrug resistant cell line by alteration of membrane biophysical properties.

Growth of CHRC5 multidrug resistant cells in media enriched in a saturated C-17 fatty acid, heptadecanoic acid, resulted in these cells accumulating vinblastine at a rate and to an extent comparable to that of the parental cell line AB1. The fatty acid-enriched growth media had no effect on the ability of AB1 cells to take up vinblastine. The action of amphiphiles on the uptake of rhodamine dyes by CHRC5 cells was compared with the increased dye accumulation affected by verapamil. Membrane rigidifying agents, such as the saturated fatty acid stearic acid, or the cholesterol derivatives, cholesteryl hemisuccinate and cholesteryl phosphorylcholine, as well as a membrane fluidizing unsaturated fatty acid, linoleic acid, could significantly increase dye uptake, although not as well as verapamil. These results taken in conjunction with other reports in the literature, demonstrate that multidrug resistance is sensitive to alterations of membrane properties. They suggest that perturbation of the membrane to either increased or to decreased membrane fluidity can lower the level of resistance.

Animals↗
PubMed1993

Calcium-independent activation of protein kinase C by the dianionic form of phosphatidic acid.

Phosphatidic acid in the form of small unilamellar vesicles has a dissociation constant of about 8.3 as determined by 31P nuclear magnetic resonance (NMR) spectroscopy. The activation of protein kinase C (PKC) by monovalent phosphatidic acid or phosphatidylserine occurs only in the presence of Ca2+. However, PKC activity on membranes of divalent anionic phosphatidic acid is independent of Ca2+ concentration.

Calcium↗
PubMed1993

Mechanism of activation of protein kinase C: roles of diolein and phosphatidylserine.

We studied the roles of lipid concentration, phosphatidylserine (PS), and diolein (DG) contents, as well as Ca2+ concentration, on the partitioning of protein kinase C (PKC) between aqueous and membrane environments as well as the relationship of this partitioning to the activation of the enzyme. Physiological concentrations of 1 mol % DG increased the apparent binding constant of PKC to the 3:1 PC/PS membrane 500 times. This increase was proportional to the mol % DG. Over 50% PKC was bound to that membrane at micromolar concentrations of Ca2+ and physiologically relevant total concentration of lipid only when 1 mol % DG was included. PKC bound either to PS alone or to PS and DG was enzymatically competent; however, the rate of phosphorylation was doubled in the presence of 1 mol % diolein. The dependence of PKC binding on the mol % PS was highly sigmoidal. The Hill coefficient was in the range of 4-6, with the higher values found at the lower lipid concentrations. These results suggest that the observed apparent cooperativity is due, at least in part, to the change in dimensionality when PKC binds to the membrane.

Animals↗
PubMed1993

Pyrazine derivatives affect membrane fluidity of vascular smooth muscle microsomes in relation to their biological activity.

We have previously demonstrated that several structurally similar pyrazine derivatives, tetramethylpyrazine (TMP), triethylpyrazine (3EP) and tetraethylpyrazine (TEP) consistently inhibited a wide spectrum of plasmalemma-associated biological activities of vascular smooth muscles in contractility and radioligand binding studies in the following order of increasing potency, TMP < 3EP < TEP. We speculated that these pyrazine derivatives elicit multiple effects in the above order of potency by affecting, at least in part, the bulk physical properties of the lipid bilayers in the cell membrane. This hypothesis was tested in the current study using plasmalemma-enriched microsomes isolated from dog aortic smooth muscle using two different biophysical techniques: fluorescence polarization and electron spin resonance. The calculated membrane fluidity values obtained as a function of temperature by both methods are consistent with the above order of potency. The amount of incorporation of these pyrazine derivatives into the vascular smooth muscle membranes also increased with the increasing hydrophobicity of these drugs, i.e., TMP < 3EP < TEP. Our results are consistent with the contention that these pyrazine derivatives not only interact with receptor protein molecules in the biological membranes, but also modulate these receptor properties via their interaction with the lipid bilayer altering the membrane microviscosity.

Animals↗
PubMed1993

Effect of end group blockage on the properties of a class A amphipathic helical peptide.

In a recent classification of biologically active amphipathic alpha-helixes, the lipid-associating domains in exchangeable plasma apolipoproteins have been classified as class A amphipathic helixes (Segrest, J.P., De Loof, H., Dohlman, J.G., Brouillette, C.G., Anantharamaiah, G.M. Proteins 8:103-117, 1990). A model peptide analog with the sequence, Asp Trp Leu Lys Ala Phe Tyr Asp Lys Val Ala Glu Lys Leu Lys Glu Ala Phe (18A), possesses the characteristics of a class A amphipathic helix. The addition of an acetyl group at the alpha-amino terminus and an amide at the alpha-carboxyl terminus, to obtain Ac-18A-NH2, produces large increases in helicity for the peptide both in solution and when associated with lipid (for 18A vs Ac-18A-NH2, from 6 to 38% helix in buffer and from 49 to 92% helix when bound to dimyristoyl phosphatidylcholine in discoidal complexes). Blocking of the end-groups of 18A stabilizes the alpha-helix in the presence of lipid by approximately 1.3 kcal/mol. There is also an increase in the self-association of the blocked peptide in aqueous solution. The free energy of binding to the PC-water interface is increased only by about 3% (from -8.0 kcal/mol for 18A to -8.3 kcal/mol for Ac-18A-NH2). The Ac-18A-NH2 has a much greater potency in raising the bilayer to hexagonal phase transition temperature of dipalmitoleoyl phosphatidylethanolamine than does 18A. In this regard Ac-18A-NH2 more closely resembles the behavior of the apolipoprotein A-I, which is the major protein component of high-density lipoprotein and a potent inhibitor of lipid hexagonal phase formation. The activation of the plasma enzyme lecithin: cholesterol acyltransferase by the Ac-18A-NH2 peptide is greater than the 18A analog and comparable to that observed with the apo A-I. In the case of Ac-18A-NH2, the higher activating potency may be due, at least in part, to the ability of the peptide to micellize egg PC vesicles.

Apolipoprotein A-I↗
PubMed1993

Role of membrane defects in the regulation of the activity of protein kinase C.

With mixtures of phosphatidylethanolamine and phosphatidylserine, the activity of protein kinase C is highly dependent on the fatty acid composition of the phosphatidylethanolamine. This contrasts with phosphatidylcholine/phosphatidylserine mixtures which affect the activity of PKC in a manner independent of the fatty acid composition of the phosphatidylcholine. The results are in accord with those phospholipids having the lowest bilayer-hexagonal phase transition temperature being most effective in augmenting the activity of PKC in the presence of its cofactors. Although the activity of this enzyme is markedly sensitive to the presence of hexagonal phase forming lipids, the activity is insensitive to differences between gel and liquid crystalline state membranes. Membrane defects alone also do not explain the observed effects since vesicles with phase boundary defects do not activate PKC. Increased hexagonal phase propensity of the lipid does not alter the partitioning of PKC between aqueous and membrane phases, which remains calcium dependent. The results demonstrate that simply the formation of defects is not sufficient to promote PKC activity, but that changes in membrane bilayer properties related to hexagonal phase propensity are required.

Animals↗
PubMed1993

Lipid concentration affects the kinetic stability of dielaidoylphosphatidylethanolamine bilayers.

The bilayer to hexagonal phase transition temperature (Th) of dielaidoylphosphatidylethanolamine is 65.5 degrees C as measured by DSC heating scans at lipid concentrations below 100 mg/ml and at scan rates ranging from 1.7 to 45 degrees C/h. However, at lipid concentrations above 100 mg/ml and at scan rates of 1-3 degrees C/h the measured Th decreases below 65.5 degrees C. At a lipid concentration of 500 mg/ml and a heating scan of 1.2 degrees C/h the transition to the hexagonal phase occurs at 62.7 degrees C. However, this same sample scanned at a rate of 34 degrees C/h has a transition temperature of 64.6 degrees C. Thus a combination of high lipid concentration and slow scan rate is required to significantly lower the hexagonal phase transition temperature below 65 degrees C. These results demonstrate that the rate of conversion of the bilayer to the hexagonal phase is dependent on the concentration of the lipid suspension even under conditions of full hydration. Furthermore, a 100 mg/ml suspension of this lipid which has a Th of 64.3 degrees C at a scan rate of 3.2 degrees C/h has a lower hexagonal phase transition temperature of 62.8 degrees C after pelleting the lipid with low-speed centrifugation but retaining the same amount of solvent in the supernatant above the pellet. Pelleting of the lipid also has a marked effect on the isothermal rate of conversion of the bilayer to hexagonal phase as observed by 31P NMR. The conversion is highly temperature-dependent and is orders of magnitude more rapid for the pelleted sample than for the suspension.(ABSTRACT TRUNCATED AT 250 WORDS)

Calorimetry, Differential Scanning↗
PubMed1993

Circular dichroism (CD) studies of antagonists derived from parathyroid hormone-related protein.

We have undertaken a study of the structure of antagonist peptides derived from the parathyroid hormone-related protein (PTHrP) in the presence of amphiphiles using circular dichroism (CD). The results were used to gain knowledge about bioactive conformations of the peptide when bound to a membrane. The substitutions within the PTHrP-(7-34)amide sequence resulted in differences in biological activity. Structural determination by CD showed the presence of an alpha-helical structure. The antagonist activity was increased in constrained peptides in which i to (i + 4) side-chain to side-chain cyclization was used to form a lactam, [Lys13,Asp17]PTHrP-(7-34)NH2. This peptide showed increased helicity in the presence of a surfactant. Hydrophobic substitutions Leu and D-Trp at positions 11 (Lys) and 12 (Gly), respectively, in PTHrP-(7-34)NH2 resulted in increased potency, but the derivatives were not significantly more helical than the unsubstituted peptide in the presence of surfactants. The combination of the hydrophobic substitutions with the constraint of lactam formation were mutually exclusive in terms of their biological activity and their alpha-helical content. We conclude that hydrophobic substitutions contribute to an increase in binding affinity by increasing hydrophobic interactions which stabilize receptor-ligand complexes. Structural rigidification, on the other hand, increases the alpha-helical content, which is important for attaining a conformation recognized by the receptor.

Circular Dichroism↗
PubMed1992

Effect of cationic cholesterol derivatives on gene transfer and protein kinase C activity.

Four different cationic derivatives of cholesterol were synthesized which contain either a tertiary or a quaternary amino head group, with and without a succinyl spacer-arm. Their ability to inhibit protein kinase C (PKC) activity was measured in a detergent mixed micellar solution. Derivatives containing a quaternary amino head group were effective inhibitors (Ki approx. 12 and 59 microM) of PKC and derivatives containing a tertiary amino head group were approx. 4-20-fold less inhibitory. Liposomes containing an equimolar mixture of dioleoylphosphatidylethanolamine (DOPE) and a cationic cholesterol derivative were tested for the DNA-mediated transfection activity in mouse L929 cells. Highest activity was found with the derivative with low PKC inhibitory activity and with a succinyl spacer-arm. The transfection activity of this tertiary amine derivative, N,N-dimethylethylenediaminyl succinyl cholesterol was dependent on DOPE as a helper lipid; liposomes containing dioleoylphosphatidylcholine and this derivative had little activity. The transfection protocol of this new cationic liposome reagent was optimized with respect to the ratio of liposome/DNA, dose of the complex and time of incubation with cells. Several adherent cell lines could be efficiently transfected with this liposome reagent without any apparent cytotoxicity. However, the transfection activity was strongly inhibited by the presence of serum components.

Animals↗
PubMed1992

Inhibition of protein kinase C by cationic amphiphiles.

A large number of PKC inhibitors are positively charged. We evaluated the structural features of cationic amphiphiles which are necessary for inhibiting PKC. Many of these compounds were derivatives of cholesterol, which possesses a hydrophobic backbone which does not perturb hydrocarbon packing in membrane bilayers. In addition, they contain a tertiary or quaternary nitrogen functionality in the head group. All designed cholesterol-based amphiphiles inhibit PKC activity; the potency of the amphiphile correlates with the presence of positive charge. Quaternary ammonium amphiphiles are 10-fold more potent than their tertiary amine counterparts, generally inhibiting in the 10-60 microM range using the Triton mixed micelle assay. Aside from charge, factors such as the structure of the amine-containing head group, its length from the hydrocarbon moiety, or the number of amine groups on the amphiphile did not markedly influence inhibitor potency. In contrast, the hydrocarbon backbone did influence potency: cationic amphiphiles containing a steroid backbone were more potent inhibitors of PKC than their straight-chain analogues. Changing the nature of the hydrocarbon from a sterol to an alkyl group lowers the pK of the amine head group so that the straight-chain analogues are no longer cationic in the conditions in the PKC assay. The results of these studies suggest that a combination of positive charge and a bilayer-stabilizing structural characteristic provides a basis for the rational design of PKC inhibitors.

Animals↗
PubMed1992

Dual modulation of protein kinase C activity by sphingosine.

Sphingosine is one of a number of cationic amphiphiles that inhibit the activity of protein kinase C (PKC) in commonly used assay conditions. This inhibition occurs only at high concentrations of this amphiphile. In the presence of excess negative charge from oleic acid, the addition of sphingosine surprisingly leads to activation of PKC. The results are explicable in terms of the dual role of charge and lipid phase propensity. When the positive charge on sphingosine is compensated by the negative charge on oleic acid, sphingosine, a hexagonal phase promoting amphiphile, becomes an activator of PKC. This does not occur with a bilayer stabilizing cationic amphiphile, N,N,N-Trimethyl-N'-cholesteryl amido-ethyl ammonium which is an inhibitor of PKC at all mol fractions, as well as in the presence of oleic acid. The results indicate that effects of sphingosine on more complex biological systems should be interpreted with caution because of this dual role of the amphiphile.

Cholesterol↗
PubMed1992

Lipid vesicles which can bind to protein kinase C and activate the enzyme in the presence of EGTA.

Maximal protein kinase C activity with vesicles of phosphatidic acid and 1,2-dioleoyl-sn-glycerol is observed in the absence of added Ca2+. Addition of phosphatidylcholine to these vesicles restores some calcium dependence of enzyme activity. 1,2-Dioleoyl-sn-glycerol eliminates the Ca(2+)-dependence of protein kinase C activity found with phosphatidic acid alone. Phorbol esters do not mimic the action of 1,2-dioleoyl-sn-glycerol in this respect. This suggests that the 1,2-dioleoyl-sn-glycerol effect is a result of changes it causes in the physical properties of the membrane rather than to specific binding to the enzyme. The effect of 1,2-dioleoyl-sn-glycerol on the phosphatidic-acid-stimulated protein kinase C activity is dependent on the molar fraction of 1,2-dioleoyl-sn-glycerol used and results in a gradual shift from Ca2+ stimulation at low 1,2-dioleoyl-sn-glycerol concentrations to calcium inhibition at higher concentrations of 1,2-dioleoyl-sn-glycerol. Phosphatidylserine-stimulated activity is also shown to be largely independent of the calcium concentration at higher molar fractions of 1,2-dioleoyl-sn-glycerol. Thus, with certain lipid compositions, protein kinase C activity becomes independent of the calcium concentration or requires only very low, stoichiometric binding of Ca2+ to high affinity sites on the enzyme. Protein kinase C can bind to phosphatidic acid vesicles more readily than it can bind to phosphatidylserine vesicles in the absence of calcium. Addition of 1,2-dioleoyl-sn-glycerol to phosphatidylserine vesicles promotes the partitioning of protein kinase C into the membrane in the absence of added Ca2+. There is no isozyme specificity in this binding. These results suggest that a less-tightly packed headgroup region of the bilayer causes increased insertion of protein kinase C into the membrane. This is a necessary but not sufficient condition for activation of the enzyme in the presence of EGTA.

Animals↗