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Biomedical subjects

R M Epand

Publications and source records attributed to R M Epand.

At least 73 records · Page 4Linked to original sources
PubMed1998

Modulation of lipid polymorphism by the feline leukemia virus fusion peptide: implications for the fusion mechanism.

The structural effects of the fusion peptide of feline leukemia virus (FeLV) on lipid polymorphism were studied, using differential scanning calorimetry (DSC), 31P nuclear magnetic resonance (NMR), and time-resolved X-ray diffraction. This peptide lowers the bilayer to inverted hexagonal phase transition temperature, TH, of dipalmitoleoylphosphatidylethanolamine (DiPoPE) at peptide mole fractions of up to 1.5 x 10(-3) at pH 5.0 and at pH 7.4. The temperature at which isotropic 31P NMR signals for monomethyldioleoylphosphatidylethanolamine (MeDOPE) first occurred is lowered by the FeLV peptide. The amount of isotropic signal seen at 40 degrees C is directly correlated to the peptide:lipid molar ratio. In the peptide-containing samples, more lipid remains in the isotropic state over the whole recorded temperature range. Isotropic 31P NMR signals were observed for DiPoPE in the presence of the FeLV peptide for the entire recorded temperature range of 35-50 degrees C, while pure DiPoPE showed no significant amount of isotropic signal. X-ray studies of DiPoPE show the formation of a new lipid phase with peptide, which is not seen in the pure lipid samples. Disordering of the Lalpha phase is evidenced by broadening of the diffraction peaks, and the hexagonal cell parameter is decreased with peptide present. Our results suggest that the FeLV peptide is increasing the negative curvature of the lipid system, which is thought to be crucial to the formation of highly bent, high-energy structural fusion intermediates, such as the "stalk" model. Fusion activity for this putative fusogenic peptide was also demonstrated, using a resonance energy transfer (RET) lipid mixing assay. To our knowledge, this work provides the first published experimental evidence of both fusogenic activity and effects on lipid polymorphism for the FeLV fusion peptide.

Amino Acid Sequence↗
PubMed1998

Increased activation of protein kinase C with cubic phase lipid compared with liposomes.

Protein kinase C (PKC) activation is measured using liposomes containing phosphatidylserine. Certain lipids display a wide range of polymorphism, depending on conditions. They can give rise to nonlamellar phases, such as hexagonal or cubic phases, as well as to lamellar phases. In this paper, we studied the activity and membrane binding of PKC in lipid bicontinuous cubic phases and hexagonal phases. The cubic phase lipid systems were (1) monoolein with 1-palmitoyl-2-oleoyl-3-phosphatidylserine (MO/PS) and (2) dielaidoylphosphatidylethanolamine/alamethicin (DEPE/alamethicin). Under fully hydrated conditions, both of the above lipid mixtures are bicontinuous cubic phases with a space group of Pn3m within certain concentration ratios and temperature ranges. Dioleoylphosphatidylethanolamine (DOPE) with up to 10 mol % PS exists in the hexagonal phase at room temperature. These cubic and hexagonal phases were able to support the PKC-catalyzed phosphorylation of histone. The amount of PKC bound to the MO/PS cubic phase showed little increase between 5 and 10 mol % PS. For both of the cubic phase systems studied, only a minor fraction of the PKC was bound to the membrane. This indicates that the specific activity of the enzyme bound to cubic phase membranes is much greater than that bound to phospholipid in the lamellar phase. Addition of up to 50 mol % MO to lipid in the lamellar phase had relatively small effects on the activity of PKC. The increase in PKC activity correlated well with an increase in PKC binding, resulting in little change in the specific activity of the membrane-bound form. These findings may be physiologically relevant due to the apparent presence of the cubic phase in certain biological structures. Also, these phases have little or no curvature strain, a property which has been shown to correlate with activation of PKC. Therefore, other factors, such as a curved morphology and/or interfacial polarity, must be responsible for the activation of PKC in these lipid systems.

Alamethicin↗
PubMed1998

Studies of kinetics and equilibrium membrane binding of class A and class L model amphipathic peptides.

We studied the kinetics and equilibrium membrane binding of two amphipathic alpha-helical peptides: the 18L peptide, which belongs to the class L (lytic peptides), and the Ac-18A-NH2 peptide of the class A (apolipoprotein), according to classification of Segrest et al. ((1990) Proteins, 8, 103-117). Both for cationic 18L and zwitterionic Ac-18A-NH2, the presence of acidic lipids increased the membrane binding constants by two orders of magnitude. The free energy of peptide-membrane association was in the range of 8.5-12.8 kcal/mol. Binding isotherms corresponded to monomer partitioning with saturation at high peptide/lipid ratios. This was also supported by stopped flow studies of the kinetics of peptide-membrane association as measured by peptide tryptophan fluorescence or by energy transfer from the peptide to the lipid-anchored anthrylvinyl fluorophor. The apparent time required for peptide-membrane equilibration was in the millisecond range. At low peptide/lipid ratios it depended on lipid concentration and was independent of the peptide concentration. The rate of peptide-membrane association was found to be relatively close to the diffusion limit. Thus peptide-membrane affinity was mostly determined by the peptide dissociation rate, i.e. higher membrane affinity correlated with a decrease in dissociation rate and with a slower peptide exchange. We have shown that the dynamic character of the peptide membrane equilibrium can be used for selective peptide targeting and disruption of membranes with a specific lipid composition.

Amino Acid Sequence↗
PubMed1998

Structural study of the interaction between the mitochondrial presequence of cytochrome c oxidase subunit IV and model membranes.

The structural effect of the presequence of cytochrome oxidase subunit IV (p25) on multilamellar liposomes with different lipid compositions has been investigated using X-ray diffraction and electron microscopy. The presequence causes the disordering of the liposomes containing negatively charged lipids, without destabilizing the bilayer structure or destroying the multilamellar nature of the liposomes. In the systems containing only zwitterionic lipids, a small increase in the d-spacing (lamellar stacking spacing) is observed without any disorder effect suggesting a weaker interaction of the peptide and lipid. Circular Dichroism measurements of the peptide, in the presence and absence of the different lipid systems studied, show that the secondary structure of the peptide is modulated by the lipid environment. Considerable amounts of alpha-helix in the presequence is only observed in the systems containing negatively charged lipids. These are the same systems for which the disordering effect is observed with X-ray diffraction. It is proposed that p25 disorders the bilayer stacking by corrugating the membranes. The results are discussed in terms of the relevance of the specific lipid properties (e.g., electric charge and ability to form inverted phases) in determining how the peptide interacts with the lipid and affects its structural organization. It is suggested that the lipid properties relevant for the disordering effect induced by the peptide are the same as those involved in the formation of contact sites between mitochondrial membranes during the import of nuclear coded proteins.

Amino Acid Sequence↗
PubMed1998

Effects of spontaneous bilayer curvature on influenza virus-mediated fusion pores.

Cells expressing the hemagglutinin protein of influenza virus were fused to planar bilayer membranes containing the fluorescent lipid probes octadecylrhodamine (R18) or indocarbocyanine (DiI) to investigate whether spontaneous curvature of each monolayer of a target membrane affects the growth of fusion pores. R18 and DiI lowered the transition temperatures for formation of an inverted hexagonal phase, indicating that these probes facilitate the formation of negative curvature structures. The probes are known to translocate from one monolayer of a bilayer membrane to the other in a voltage-dependent manner. The spontaneous curvature of the cis monolayer (facing the cells) or the trans monolayer could therefore be made more negative through control of the polarity of voltage across the planar membrane. Electrical admittance measurements showed that the open times of flickering fusion pores were shorter when probes were in trans monolayers and longer when in cis monolayers compared with times when probe was symmetrically distributed. Open times were the same for probe symmetrically distributed as when probes were not present. Thus, open times were a function of the asymmetry of the spontaneous curvature between the trans and cis monolayers. Enriching the cis monolayer with a negative curvature probe reduced the probability that a small pore would fully enlarge, whereas enriching the trans monolayer promoted enlargement. Lysophosphatidylcholine has positive spontaneous curvature and does not translocate. When lysophosphatidylcholine was placed in trans leaflets of planar membranes, closing of fusion pores was rare. The effects of the negative and positive spontaneous curvature probes do not support the hypothesis that a flickering pore closes from an open state within a hemifusion diaphragm (essentially a "flat" structure). Rather, such effects support the hypothesis that the membrane surrounding the open pore forms a three-dimensional hourglass shape from which the pore flickers shut.

3T3 Cells↗
PubMed1997

Influence of the angle subtended by the positively charged helix face on the membrane activity of amphipathic, antibacterial peptides.

To investigate the influence of the angle subtended by the positively charged helix face on membrane activity, six amphipathic alpha-helical peptides with angles between 80 degrees and 180 degrees, but with retained hydrophobicity, hydrophobic moment, and positive overall charge, were designed starting from the sequence of the antibacterial peptide magainin 2. CD investigations revealed that all analogs are in an alpha-helical conformation in vesicle suspension. The ability of the peptides to induce dye release from negatively charged phosphatidylglycerol (PG) vesicles decreased with increasing angle. However, peptides with a large angle of positively charged residues (140-180 degrees) exhibited a considerably higher permeabilizing activity at zwitterionic phosphatidylcholine (PC) and mixed PC/PG (3:1) vesicles than analogs with a small angle (80-120 degrees). In addition, analogs with large angles were more active in antibacterial and hemolytic assays. The antibacterial specificity of these analogs was decreased. Binding investigations showed that peptide binding is favored by a large angle and a high content of negatively charged phospholipid. In contrast, a small angle and a low negative membrane charge enhanced the membrane-permeabilizing efficiency of the bound peptide fraction. All analogs stabilized the bilayer phase of phosphatidylethanolamine over the inverted hexagonal phase. Therefore, a class L mechanism of permeabilization can be excluded. Furthermore, the analogs do not act by the induction of positive curvature strain or by a "carpet-like" mechanism. Our results are in accordance with a pore mechanism: The membrane-permeabilizing efficiency of analogs with enhanced angle of positively charged residues is reduced due to electrostatic repulsion between adjacent helices within the pore, thus resulting in a decreased pore-forming probability and/or pore destabilization.

Amino Acid Sequence↗
PubMed1997

Structural aspects of the interaction of peptidyl-glycylleucine-carboxyamide, a highly potent antimicrobial peptide from frog skin, with lipids.

The interaction of PGLa (peptidyl-glycylleucine-carboxyamide), a 21-amino-acid residue cationic peptide, isolated from the skin of the South African clawed frog, Xenopus laevis, with model membrane systems was investigated. Our studies focussed on the importance of the difference in the phospholipid composition of bacterial and erythrocyte membranes. This is of particular interest to gain information on the specificity of membranolysis exhibited by this peptide against bacteria but not against erythrocytes. In phosphate buffer at physiological pH, as well as in the presence of the zwitterionic phosphatidylcholine and sphingomyelin. the peptide had a random structure but it adopted an alpha-helical conformation in the presence of negatively charged lipids. Furthermore, calorimetric experiments showed that PGLa had no effects on the thermotropic phase behavior of liposomes composed of the choline phosphatides, while separation of a distinct peptide-rich domain was observed for phosphatidylglycerol liposomes. In addition to the main transition of pure 1,2-dipalmitoylglycerophosphoglycerol at 40 degrees C a second transition owing to the peptide-perturbed lipid domains was found at 41 degrees C. This conclusion is supported by X-ray diffraction experiments which indicated that PGLa penetrates into the hydrophobic core of the bilayer inducing an untilting of the hydrocarbon chains as observed in the gel phase of the pure lipid. These results demonstrate that this antibacterial peptide specifically interacts with negatively charged lipid membranes, which are characteristic of bacterial membranes. This can be explained based on the structural features of PGLa.

Animals↗
PubMed1997

Amphipathic peptide affects the lateral domain organization of lipid bilayers.

Using lipid-specific fluorescent probes, we studied the effects of amphipathic helical, membrane active peptides of the A- and L-type on membrane domain organization. In zwitterionic binary systems composed of mixtures of phosphatidylcholine and phosphatidylethanolamine, both types of peptides associated with the fluid phase. While binding with high affinity to fluid membranes, peptides were unable to penetrate into the lipid membrane in the gel state. If trapped kinetically by cooling from the fluid phase, peptides dissociated from the gel membrane on the time scale of several hours. While the geometrical shape of the alpha-helical peptides determines their interactions with membranes with non-bilayer phase propensity, the shape complementarity mechanism by itself is unable to induce lateral phase separation in a fluid membrane. Charge-charge interactions are capable of inducing lateral domain formation in fluid membranes. Both peptides had affinity for anionic lipids which resulted in about 30% enrichment of acidic lipids within several nanometers of the peptide's tryptophan, but there was no long-range order in peptide-induced lipid demixing. Peptide insertion in fluid acidic membranes was accompanied by only a small increase in bilayer surface and a decrease in polarity in the membrane core. Peptide-lipid charge-charge interactions were also capable of modulating existing domain composition in the course of the main phase transition in mixtures of anionic phosphatidylglycerol with zwitterionic phosphatidylcholine.

Amino Acid Sequence↗
PubMed1997

Role of lipids in the permeabilization of membranes by class L amphipathic helical peptides.

We studied the mechanism of membrane permeabilization by the 18L model peptide (GIKKFLGSIWKFIKAFVG), which features the consensus class L sequence averaged from the number of naturally occurring lytic peptides. Two aspects of membrane lipid composition significantly affected peptide-membrane interactions: the presence of acidic lipids and, in zwitterionic membranes, and the presence of nonbilayer forming lipids. In zwitterionic membranes, 18L peptide destabilizes the membrane, leading to a transient formation of large defects in the membrane which result generally in contents leakage, but in the presence of bilayer-bilayer contact can alternatively lead to vesicle fusion. In membranes containing acidic lipids (DOPC:DOPG, DOPG), 18L caused leakage but not fusion, probably due to mutual repulsion of acidic vesicles. While the extent of contents leakage was approximately the same as for zwitterionic membranes, the kinetics of leakage could be resolved only by using stopped-flow, leakage being essentially complete within the first minute. Previously, we reported that apolipoprotein (class A) and lytic (class L) peptide analogs have opposing effects on some properties of biological membranes. This reciprocal effect of 18L and Ac-18A-NH2, class A model peptide, is restricted to membranes with a high propensity for nonbilayer phase formation (DOPE, Me-DOPE, DOPC:DOPE, DOPC:Me-DOPE). The decrease in the content of nonbilayer phase forming lipid or the addition of acidic lipids reduces or eliminates the reciprocal effects. This suggests the importance of nonbilayer phase propensity for certain functions of biological membranes.

Amino Acid Sequence↗
PubMed1997

Lipogastrins as potent inhibitors of viral fusion.

The rate and extent of membrane fusion is markedly sensitive to membrane interfacial properties. Lipopeptides with hydrophilic peptide moieties will insert into membranes, leaving the peptide portion at the membrane-water interface. In this work, we have used a lipopeptide composed of the peptide [Nle15]-gastrin-(2-17)-amide covalently linked to 1,2-diacyl-3-mercaptoglycerol-N(alpha)-maleoyl-beta-alanine to give DM-gastrin or DP-gastrin having 14 or 16 carbon atom acyl chains, respectively. The fluorescence emission from the two Trp residues of these lipopeptides exhibited little or no blue shift upon addition of liposomes of egg-phosphatidylethanolamine containing 5 mol% G(D1a). Iodide quenching of DP-gastrin fluorescence was also independent of lipid. These results indicate that the peptide moiety is exposed to the aqueous environment even though the lipopeptide is firmly anchored to the membrane. Both DM and DP-gastrin markedly raise the bilayer to hexagonal phase transition temperature of dipalmitoleoyl phosphatidylethanolamine. However, DM-E5 lowers this phase transition temperature. These lipopeptides have effects on the overall fusion of Sendai virus to liposomes in accord with their opposite effects on lipid curvature. The lipogastrins are potent inhibitors of viral fusion, while DM-E5 slightly promotes this process. Truncated forms of DM-gastrin are also inhibitory to viral fusion, but are less inhibitory than the full lipopeptide. Analysis of the fusion kinetics shows that DP-gastrin causes a reduction in the final extent of fusion and a marked lowering of the fusion rate constant. Binding of Sendai virus to the ganglioside receptor-containing liposomes was not affected. Consideration of the various contributions to the mechanism of inhibition of viral fusion suggests that effects of lipogastrin on membrane intrinsic monolayer curvature is of primary importance.

Amino Acid Sequence↗
PubMed1997

Structural study of the relationship between the rate of membrane fusion and the ability of the fusion peptide of influenza virus to perturb bilayers.

The amino-terminal segment of the HA2 protein of influenza virus (fusion peptide) has been identified as an important region for membrane fusion. The wild type virus can fuse to membranes more rapidly at pH 5 than at pH 7.4. It has been demonstrated that there is a relationship between the ability of the peptide to promote the formation of inverted phases and the fusogenicity of the intact virus. In this work, we use small-angle X-ray diffraction to study the mechanism of the structural effect of the peptide, at different pHs, on lipid systems characterized by each having a different spontaneous radius of curvature. The overall results show that the action of the peptide on the polymorphism of the lipid systems investigated is strongly pH-dependent. In particular, a rapid formation of cubic phases at pH 5.0 is observed in the presence of this fusion peptide. The ability of the fusion peptide to promote cubic phases exhibits the same dependence on the pH as does the fusogenicity of the intact virus. It is proposed that the peptide promotes cubic phases at pH 5.0 by changing the kinetics of the lamellar to inverted phase transitions.

Amino Acid Sequence↗
PubMed1997

Role of water in protein kinase C catalysis and its binding to membranes.

The role of hydration in the catalytic activity and membrane binding of rat brain protein kinase C (PKC) was investigated by modulating the activity of water with polyethylene glycols with molecular weights of 1000-20000 and dextran with a molecular weight of 20000. These polymers create an osmotic stress due to their exclusion from hydration shells and crevices on proteins, causing dehydration. Polymers larger than 1000 caused an activation of the PKC-catalyzed phosphorylation of histone, while PEG 1000 had no significant effect. The extent of activation by PEG and dextran 20000 was larger than that of PEG 6000 or 8000 when vesicles were composed of 1:1 POPS/POPC, suggesting the presence of at least two distinct regions of exclusion on PKC: one inaccessible to PEGs larger than 1000 and the other inaccessible only to PEGs of > 10000. The extent of activation was dependent on the composition of the vesicles used. If basal activity (without PEG) was low (e.g. with low PS content in membranes), then the extent of activation was similar for all polymers larger than 1000. Binding of PKC to membranes containing 50 mol % PS was unaffected by PEG 6000 but was inhibited by PEG 20000. At a low PS content of 10%, both PEG 6000 and 20000 inhibited binding. This suggests that PKC becomes hydrated upon binding to membranes. Under conditions in which all of the enzyme is membrane-bound, both Km and Vmax for the phosphorylation of histone increased linearly with osmotic stress induced by PEG 6000. Thus, PKC becomes hydrated with 2311 +/- 476 water molecules upon binding of histone and is dehydrated by 1349 +/- 882 water molecules in going to the transition state. Km and Vmax for phosphorylation of the MARCKS peptide also increase with osmotic stress induced by PEG 6000. When protamine sulfate was used as a substrate (cofactor-independent), Vmax for the reaction was unaffected, but Km decreased with osmotic pressure (with PEG 6000), suggesting that PKC becomes dehydrated upon binding protamine. Similar results were found with a peptide substrate derived from the pseudosubstrate site of PKC epsilon. Since dextran, a polymer unrelated in structure to PEG, could cause a similar activation of PKC, the effects seen are likely due to osmotic stress and not to specific binding of PEG to PKC. Also, results obtained with PE-linked PEG were opposite to those with free PEG. PE-linked PEGs of 2000 and 5000 caused an inhibition of PKC-catalyzed phosphorylation of histone when present in membranes. If a specific interaction occurred with PEG, this would be expected to occur even with PE-PEG. The effects observed with free PEG are also independent of ionic strength. Free PEG had no effect on the bilayer to hexagonal phase transition temperature of DEPE membranes, suggesting that the effects on PKC activity are not a consequence of changes in membrane properties at the osmotic pressures used.

Amino Acid Sequence↗
PubMed1997

Biophysical studies of lipopeptide-membrane interactions.

There are a number of naturally occurring motifs for lipidation of peptides and proteins. In cases in which this involves adding a single hydrocarbon chain to the peptide, it is either a fatty acid or an isoprenyl group. Lipopeptides will partition between membrane and aqueous phases. When only one hydrocarbon chain is attached to the peptide, the affinity of the lipopeptide for the membrane is only marginally increased over that of the free peptide. The resulting partitioning is largely determined by the extent of the interaction of the peptide moiety with the membrane. In contrast, lipidation involving two hydrocarbon chains, either as two single chains attached at distinct locations of the peptide or a double-chain lipid anchor, firmly attaches the lipopeptide to the membrane. This can allow the placement of specific binding sites on a membrane surface. Such a strategy can be used, for example, to place specific antibodies on the surface of drug-carrying liposomes for the purpose of targeting drug delivery. In addition, lipopeptides will alter the physical properties of membranes. One of these effects is to increase the bilayer to hexagonal phase transition temperature. Substances having this property may also alter functional properties of membranes. While it is unlikely that these changes in the biophysical properties of the membranes. While it is unlikely that these changes in the biophysical properties of the membrane are responsible for specific functions of lipopeptides, such changes may be used to modulate certain properties of a membrane, such as the rate of viral fusion.

Biophysical Phenomena↗
PubMed1997

The mechanism of lamellar-to-inverted hexagonal phase transitions in phosphatidylethanolamine: implications for membrane fusion mechanisms.

We studied the mechanism of the lamellar-to-inverted hexagonal (L alpha/H[II]) phase transition, using time-resolved cryotransmission electron microscopy (TRC-TEM), 31P-NMR, and differential scanning calorimetry. The transition was initiated in dispersions of large unilamellar vesicles of dipalmitoleoyl phosphatidylethanolamine (DiPoPE). We present evidence that the transition proceeds in three steps. First, many small connections form between apposed membranes. Second, the connections aggregate within the planes of the bilayers, forming arrays with hexagonal order in some projections. Third, these quasihexagonal structures elongate into small domains of H(II) phase, acquiring lipid molecules by diffusion from contiguous bilayers. A previously proposed membrane fusion mechanism rationalizes these results. The modified stalk theory predicts that the L alpha/H(II) phase transition involves some of the same intermediate structures as membrane fusion. The small interbilayer connections observed via TRC-TEM are compatible with the structure of a critical intermediate in the modified stalk mechanism: the trans monolayer contact (TMC). The theory predicts that 1) TMCs should form starting at tens of degrees below TH; 2) when TMCs become sufficiently numerous, they should aggregate into transient arrays like the quasihexagonal arrays observed here by TRC-TEM; and 3) these quasihexagonal arrays can then elongate directly into H(II) phase domains. These predictions rationalize the principal features of our data, which are incompatible with the other transition mechanisms proposed to date. Thus these results support the modified stalk mechanism for both membrane fusion and the L alpha/H(II) phase transition. We also discuss some implications of the modified stalk theory for fusion in protein-containing systems. Specifically, we point out that recent data on the effects of hydrophobic peptides and viral fusion peptides on lipid phase behavior are consistent with an effect of the peptides on TMC stability.

Biophysical Phenomena↗
PubMed1997

Effect of N-acyl-phosphatidylethanolamine on the membrane fusion between Sendai virus and liposome.

We have compared the properties of two N-acyl derivatives of dilauryl phosphatidylethanolamine on lipid polymorphism, vesicle leakage and Sendai virus fusion. The derivatives contained either an N-lauroyl group (NLPE) or an N-acetyl group (NAcPE). Only the NAcPE markedly affected the bilayer to hexagonal transition temperature of dielaidoyl phosphatidylethanolamine, shifting it to higher values. In contrast the NLPE slightly lowered this phase transition temperature. The two lipids also have opposite effects on leakage from small unilamellar vesicles of egg phosphatidylcholine. The NLPE inhibits leakage, while the NAcPE promotes it. This vesicle stabilizing effect of NLPE against leakage is not manifested in alterations of rates or extents of Sendai virus fusion to liposomes of egg phosphatidylethanolamine plus 2% ganglioside GD1a. The NLPE has no effect, while the NAcPE reduces the observed fusion, at least in part as a consequence of a reduction in the final extent of fusion. These results demonstrate that the bilayer stabilizing effects of NLPE do not result in a lower rate of viral fusion. Furthermore, these bilayer stabilizing effects against leakage are not solely a function of the lipid headgroup but also require a structure with three long acyl chains. The reduced leakage is not related to a loss in monolayer curvature strain.

Acylation↗
PubMed1996

Fluorescent probes of membrane surface properties.

We have studied the properties of two new fluorescent probes, 7-dimethylaminocoumarin derivatives, 4-[N, N-dimethyl-N-(n-tetradecyl)ammoniummethyl]-7-(N,N-dimethylamino)co umarin chloride (TAMAC) and 4-(n-dodecylthiomethyl)-7-(N,N-dimethylamino)coumarin (DTMAC) in model membrane systems. Both probes are sensitive to solvent polarity. The TAMAC probe has a quaternary ammonium function to position it at a fixed location with respect to the membrane interface. In membranes of dipalmitoleoylphosphatidylethanolamine (DiPoPE), both probes detect marked increases in surface hydrophobicity as the bilayer to hexagonal phase transition temperature is approached. This does not occur when the probes are embedded in dipalmitoleoylphosphatidylcholine (DiPoPC) in which case the fluorescence emission is found to be largely independent of temperature. A nitroxide quencher covalently linked to the 5 position of the sn-2 acyl chain of phosphatidylcholine quenches the fluorescence of DTMAC in DiPoPC more than in DiPoPE, indicating the deeper insertion of this probe in DiPoPC. As the temperature is increased the DTMAC fluorophore moves even further out of the membrane. These findings indicate that DTMAC, which does not contain a group to fix its location along the bilayer normal, adjusts its position to small changes in environment polarity, so as to maintain an environment of a fixed dielectric constant. However, with greater changes in membrane interfacial polarity the environment of the probe will be altered. Thus, in addition to the sensitivity of these probes to solvent polarity, the ability of a fixed nitroxide to quench DTMAC becomes another parameter with which to characterize membrane properties with these probes.

Coumarins↗
PubMed1996

Transbilayer inhibition of protein kinase C by the lipophosphoglycan from Leishmania donovani.

Lipophosphoglycan (LPG), the predominant molecule on the surface of the parasite Leishmania donovani, has previously been shown to be a potent inhibitor of protein kinase C (PKC) isolated from rat brain. The mechanism by which LPG inhibits PKC was further investigated in this study. LPG was found to inhibit the PKC alpha-catalyzed phosphorylation of histone in assays using large unilamellar vesicles composed of 1-palmitoyl, 2-oleoyl phosphatidylserine and 1-palmitoyl, 2-oleoyl phosphatidylcholine either with or without 1% 1,2 diolein added. The results also indicated that while PKC binding to sucrose-loaded vesicles was not substantially reduced in the presence of LPG at concentrations of 1-2%, the activity of membrane-bound PKC was inhibited by 70%. This inhibition of the membrane-bound form of PKC is not a consequence of reduced substrate availability to the membrane. However, Km shifted from approximately 31 +/- 4 microM to 105 +/- 26 microM in the presence of 5% LPG. LPG caused PKC to bind to membranes without inducing a conformational change as revealed by the lack of an increased susceptibility to trypsin. An LPG fragment containing only one repeating disaccharide unit was not as effective as the entire LPG molecule or of larger fragments in inhibiting the membrane-bound form of the enzyme. The shorter fragments were also less potent in raising the bilayer to hexagonal phase transition temperature of a model membrane. LPG is also able to inhibit the membrane-bound form of PKC alpha from the inner monolayer of large unilamellar vesicles, the opposite monolayer to which the enzyme binds in our assay. Inhibition is likely a result of alterations in the physical properties of the membrane. To our knowledge, this is the first example of a membrane additive that can inhibit the membrane-bound form of PKC in the presence of other lipid cofactors.

Amino Acid Sequence↗