Presence Of Dimyristoylphosphatidylcholine Research Articles

Stable Small Composite Microbubbles Decorated with Magnetite Nanoparticles - A Synergistic Effect between Surfactant Molecules and Nanoparticles.

Three approaches to preparing iron oxide nanoparticle-decorated microbubbles (NP-decoMBs) have been investigated. The size and stability characteristics of these microbubbles (MBs) were investigated by optical microscopy, laser light scattering and an acoustical method, and compared with those of non-decorated MBs. First, magnetite nanoparticles (Fe3O4NPs) grafted with dimyristoylphosphatidylcholine (DMPC) were synthesized and used to prepare MBs by brief sonication under an atmosphere of air saturated with perfluorohexane. These MBs had a rather large mean radius (r ~ 12 µm), and a moderate volume of encapsulated gas. Remarkably, a second approach that consisted of dispersing unbound DMPC molecules in the aqueous phase along with DMPC-grafted Fe3O4NPs prior to sonication was found to drastically change the situation, allowing the obtaining of monomodal populations of much smaller (r ~ 0.6 µm) NP-decoMBs. The latter were echogenic and stable for at least 10 days at room temperature, without significant variation of their size characteristics. In a third approach, NP-decoMBs were directly prepared from dispersions of naked Fe3O4NPs in the presence of DMPC. The resulting NP-decoMBs suspensions consisted of broadly distributed bubble populations mostly containing two populations (with r ~ 5 and ~ 15 µm). Control microbubbles made of DMPC only were small (r ~ 1.3 µm), although not as small as those formed from DMPC-grafted Fe3O4NPs in the presence of free DMPC, and were less stable, with a room temperature half-life of only ~1 day. These observations imply that there is a synergy between the Fe3O4NPs and the DMPC molecules in the air/water interfacial film stabilization process.

Membrane defects enhance the interaction of antimicrobial peptides, aurein 1.2 versus caerin 1.1

The membrane interactions of the antimicrobial peptides aurein 1.2 and caerin 1.1 were observed by 31P and 2H solid-state NMR and circular dichroism spectroscopy. Both peptides were relatively unstructured in water. In the presence of dimyristoylphosphatidylcholine (DMPC) and mixed DMPC and dimyristoylphosphatidylglycerol (DMPG) vesicles, both peptides displayed a considerable increase in helical content with the shorter aurein peptide having a higher α-helix content in both lipid systems. In fluid phase DMPC vesicles, the peptides displayed differential interactions: aurein 1.2 interacted primarily with the bilayer surface, while the longer caerin 1.1 was able to penetrate into the bilayer interior. Both peptides displayed a preferential interaction with the DMPG component in DMPC/DMPG bilayers, with aurein 1.2 limited to interaction with the surface and caerin 1.1 able to penetrate into the bilayer and promote formation of a mixture of lipid phases or domains. In gel phase DMPC vesicles, aurein 1.2 disrupted the bilayer apparently through a carpet mechanism, while no additional interaction was seen with caerin 1.1. Although a lamellar bilayer was retained with the mixed DMPC/DMPG vesicles below the phase transition, both caerin 1.1 and aurein 1.2 promoted disruption of the bilayer and formation of an isotropic phase. The peptide interaction was enhanced relative to the fluid phase and was likely driven by co-existence of membrane defects. This study thus demonstrates that the effects of the lipid phase and domains need to be considered when studying membrane interactions of antimicrobial peptides.

Effect of salt on the interaction of Hal18 with lipid membranes

One of the major obstacles in the development of new antimicrobial peptides as novel antibiotics is salt sensitivity. Hal18, an α-helical subunit of Halocidin isolated from Halocynthia aurantium, has been previously shown to maintain its antimicrobial activity in high salt conditions. The α-helicity of Hal18 in the presence and absence of salt was demonstrated by circular dichroism spectroscopy, which showed that the peptide was mainly unordered containing β-strands and β-turns. However, in the presence of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylserine (DMPS) vesicles, Hal18 folded to form α-helices (circa 42%). Furthermore, the structure was not significantly affected by pH or the presence of metal ions. These data were supported by monolayer results showing Hal18 induced stable surface pressure changes in monolayers composed of DMPC (5mNm(-1)) and DMPS (8.5mNm(-1)), which again were not effected by the presence of metal ions or pH. It is proposed that the hydrophobic groove within its molecular architecture enables the peptide to form stable associations with lipid membranes. The balance of hydrophobicity along the Hal18 long axis would also support oblique orientation of the peptide at the membrane interface. Hence, this model of membrane interaction would enable the peptide to penetrate deep into the membrane. This concept is supported by lysis data. Overall, it would appear that this peptide is a potential candidate for future AMP design for use in high salt environments.

Ortho -quinone-enhanced ascorbate oxidation. Combined roles of lipid charge and the magnesium cation

Quinones are widely distributed compounds in nature. Of these, ortho-quinones are found to be involved in the pathogenic mechanism of Parkinson's disease, in oxidative deaminations to free-radical redox reactions, and as intermediates in the pathways implicated in the carcinogenicity of 2,3- and 3,4-catechol estrogens. Addition of MgCl2 to solutions of the hydrophobic ortho-quinones, 1,10-phenanthroquinone (PHQ) and beta-lapachone (LQ) enhances ascorbate oxidation in the absence or presence of large unilamellar vesicles (LUVs) of the neutral lipid dimyristoylphosphatidylcholine (DMPC), although initial rates of ascorbate oxidation are smaller in the presence of lipid as compared to its absence. Addition of this salt to solutions of the para-quinone 1,4-naphthoquinone (NQ) did not affect the ascorbate rate of oxidation in the absence or presence of DMPC. Addition of MgCl2 to semiquinone solutions of PHQ or LQ in the presence or absence of DMPC increases semiquinone stability, as detected from the semiquinone disproportionation equilibrium displacement to semiquinone formation. Furthermore, MgCl2 increases the partition of the ortho-semiquinones into the aqueous phase, although no such effect is observed for the semiquinone of NQ. For all the quinones under study, smaller rates of ascorbate oxidation and of semiquinone equilibrium concentration occur in the presence of negatively charged LUVs composed of an equimolar mixture of DMPC and dimyristoylphosphatidic acid DMPA. Ascorbate oxidation rate enhancements correlate with an increase in semiquinone concentration with addition of MgCl2, in the absence or presence of neutral lipid. This observation favors the proposition that ascorbate oxidation rate increases are caused by semiquinone thermodynamic stabilization. Thus, the ascorbate oxidation rate enhancement by MgCl2 in solutions containing hydrophobic ortho-quinones is still possible in systems with hydrophobic environments analogous to that of DMPC.

Some Insights about 1,6-Diphenyl-1,3,5-hexatriene—Lipid Supramolecular Assemblies by Steady-State Fluorescence Measurements

1,6-Diphenyl-1,3,5-hexatriene (DPH) is the most widely proposed molecular probe for the post-column fluorescence derivatization of lipids after liquid chromatography separation. This kind of detection consists of a supramolecular combination of DPH and eluted lipids. The detection is optimally performed in a mainly aqueous environment (over 80% v/v) because the weak fluorescence of DPH in water is drastically enhanced upon formation of supramolecular assemblies with lipids. In the present study, and in order to obtain better spectroscopic insights into the nature of these supramolecular assemblies, two different lipids were tested, 1,2,3-tridodecanoylglycerol (LLL) as a model triglyceride (nonpolar lipid) and dimyristoylphosphatidylcholine (DMPC) as a model phosphatidylcholine (charged amphiphilic lipid). Stoichiometry and association constants were determined on the basis of the variation of fluorescence intensity in the presence of various concentrations of lipids. LLL(60)-DPH(2) and DMPC(200)-DPH(2) complexes were identified with association constants as high as K(2) = (5.8 +/- 0.5) x 10(13) M(-2) and (17.3 +/- 2.0) x 10(13) M(-2) for LLL and DMPC, respectively. The fluorescence intensity of DPH in the presence of LLL is greater than in the presence of DMPC. An attempt to characterize the insertion mode of DPH in the lipidic supramolecular assemblies is also made.

Two-dimensional crystallization of human vitamin K-dependent γ-glutamyl carboxylase

Planar-tubular two-dimensional (2D) crystals of human vitamin K-dependent γ-glutamyl carboxylase grow in the presence of dimyristoyl phosphatidylcholine (DMPC). Surprisingly, these crystals form below the phase transition temperature of DMPC and at the unusually low molar lipid-to-protein (LPR) ratio of 1, while 2D crystals are conventionally grown above the phase transition temperature of the reconstituting lipid and significantly higher LPRs. The crystals are up to 0.75 μm in the shorter dimension of the planar tubes and at least 1 μm in length. Due to the planar-tubular nature of the crystals, two lattices are present. These are rotated by nearly 90° in respect to each other. The ordered arrays exhibit p12 1 plane group symmetry with unit cell dimensions of a = 83.7 Å, b = 76.6 Å, γ = 91°. Projection maps calculated from images of negatively stained and electron cryo-microscopy samples reveal the human vitamin K-dependent γ-glutamyl carboxylase to be a monomer.

Solvation Dynamics in Dimyristoyl-Phosphatidylcholine Entrapped Inside a Sol−Gel Matrix

Solvation dynamics of coumarin 480 (C480) is studied in a sol−gel matrix containing dimyristoyl-phosphatidylcholine (DMPC). In the presence of DMPC, the average solvation time (〈τs〉) in the sol−gel glass is found to be 500 ps. This indicates that in the sol−gel matrix solvation dynamics in the presence of DMPC is slower than that in its absence. The solvation dynamics of C480 in the DMPC entrapped TEOS sol−gel matrix is much faster than that in DMPC liposomes in bulk water. This suggests that the DMPC entrapped TEOS sol−gel matrix is very different from DMPC liposomes in bulk water. The rotational relaxation of C480 is found to be very fast giving rise to a very small initial anisotropy value (r0 = 0.08). This shows that even after incorporation of lipids inside the TEOS sol−gel matrix, the probe C480 remains highly mobile.

A synthetic peptide that inhibits lipoprotein(a) assembly.

We previously reported that human apolipoprotein B100 (apoB) amino acids 4330-4397 were important for the initial noncovalent binding to apolipoprotein(a) [apo(a)] that facilitates lipoprotein(a) [Lp(a)] assembly. In this study, we aimed to further define the apoB sequences within the 4330-4397 region that were important for the noncovalent binding to apo(a). Alignment of the human apoB4330-4397 sequence with mouse apoB, which also noncovalently binds apo(a), revealed stretches of similar sequence, including a lysine-rich sequence spanning apoB amino acids 4372-4392. Structural analysis of the apoB4372-4392 sequence using the WHEEL program predicted an amphipathic alpha-helix. Circular dichroism studies of a synthetic peptide spanning human apoB amino acids 4372-4392, both in the absence and presence of dimyristoylphosphatidylcholine, confirmed the alpha-helical nature of the sequence. We tested the ability of the apoB4372-4392 peptide to bind to apo(a) and found that the peptide bound to apo(a) with high affinity but not to Lp(a). The apoB4372-4392 peptide inhibited Lp(a) assembly in Lp(a) formation assays far more effectively than the lysine analogue, epsilon-amino-n-caproic acid (IC50=40 micromol/L versus 10 mmol/L, respectively). Incorporation of the apoB4372-4392 peptide onto dimyristoylphosphatidylcholine vesicles yielded an even more effective inhibitor (IC50=4 micromol/L). Our study shows that the apoB4372-4392 sequence mediates the initial noncovalent binding to apo(a) and has demonstrated that the apoB4372-4392 peptide is a novel and effective inhibitor of Lp(a) assembly.

EPR study of β-phosphorylated stable aminoxyl radicals in the presence of liposomes

The behaviour of four stable β-phosphorylated aminoxyl radicals of the pyrrolidinoxyl series has been studied by EPR spectroscopy in the presence of dimyristoyl-phosphatidylcholine (DMPC) unilamellar liposomes. The affinity of these compounds for the liposome structure was found to be more or less high depending on their hydrophobicity. In each case, computer simulations of experimental EPR spectra permitted the determination of the hyperfine coupling constants of the radical inserted in the phospholipid bilayer.

Insect immune activation by apolipophorin III is correlated with the lipid-binding properties of this protein.

Apolipophorin III (apoLp-III) is an exchangeable insect apolipoprotein consisting of five amphipathic alpha-helices. The protein is able to open reversibly on associating with hydrophobic surfaces and plays a role both in lipid transport and induction of immune responses. Point mutations were introduced at positions 66 (N-->D) and/or 68 (K-->E) between helices 2 and 3, a region possibly serving as a hinge for the opening of the molecule when associating with lipids. The lipid-binding properties of the mutant proteins were analyzed and compared with their immune inducing activities. Structural properties of the proteins were studied by far UV circular dichroism spectroscopy and their abilities to form discoidal complexes of dimyristoyl phosphatidylcholine (DMPC) vesicles were investigated. In comparison to wild-type apoLp-III, apoLp-III(N66D/K68E), and apoLp-III(K68E) displayed significantly decreased lipid-binding abilities and immune stimulating activities, while these effects were less noticeable with apoLp-III(N66D). The secondary structure of the double mutant apoLp-III(N66D/K68E) was similar to that of wild-type apoLp-III. A noticeable reduction of alpha-helical content could be observed for the single mutants apoLp-III(N66D) and apoLp-III(K68E), which was accompanied by an increase in percentage amount of beta-turns. The stability of the secondary structure determined by heat denaturation was not affected by mutagenesis. Furthermore, the ability of all proteins to form discoidal complexes of equal size and shape in the presence of dimyristoyl phosphatidylcholine indicated that the mutagenesis did not affect the molecular architecture in the lipid-associated conformation. The relationship between reduced lipid association and reduced immune stimulating activity supports the hypothesis that apoLp-III-induced immune activation is triggered by the conformational change of the protein.

Crystallization of the Human, Mitochondrial Voltage-Dependent Anion-Selective Channel in the Presence of Phospholipids

Overexpressed human voltage-dependent anion-selective channel VDAC or porin from mitochondrial outer membranes has been purified to homogeneity. Electron microscopic analysis of VDAC in detergent solution revealed a uniform particle population consisting of porin monomers. After dialysis of detergent-solubilized porin in the presence of dimyristoylphosphatidylcholine at lipid-to-protein ratios between 0.2 and 0.5 (percentage by weight), mostly multilamellar crystals were obtained. Crystals adsorbed to carbon films flattened during negative staining and air-drying and exhibited different structural features due to differences in the vertical stacking of several crystalline layers, each consisting of one membrane bilayer. Adsorbed, frozen-hydrated multilamellar membrane crystals revealed uniform diffraction patterns with sharp diffraction spots extending to 8.2 Å. The surface structure of VDAC was reconstructed from freeze-dried and unidirectionally metal-shadowed crystals. Major protein protrusions were observed from two VDAC monomers present in the unit cell. Differences in the surface structural features indicate alternate orientations of VDAC molecules with respect to the lipid bilayer, allowing the simultaneous imaging of both the cytosolic and intramitochondrial surfaces. Each VDAC molecule consists of a pore lumen with a diameter of 17–20 Å surrounded by a protein rim of nonuniform height, suggesting an asymmetrical distribution of protein mass around the diffusion channels.

Conformation of Two Peptides Corresponding to Human Apolipoprotein C-I Residues 7-24 and 35-53 in the Presence of Sodium Dodecyl Sulfate by CD and NMR Spectroscopy

Peptides corresponding to the proposed lipid-binding domains of human apolipoprotein C-I, residues 7-24 (ALDKLKEFGNTLEDKARE) and 35-53 (SAKMREWFSETFQKVKEKL), were studied by CD and two-dimensional 1H NMR spectroscopy. Sodium dodecyl sulfate (SDS) was used to model the lipoprotein environment. Analysis of the CD data shows that both peptides lack well-defined structure in aqueous solution but adopt helical, ordered structures upon the addition of SDS. The helical nature of the peptides in the presence of SDS was confirmed by H alpha secondary shifts. A total of 199 (apoC-I(7-24)) and 266 (apoC-I(35-53)) distance restraints were used in distance geometry and simulated annealing calculations to generate average structures for both peptides in aqueous solutions containing SDS. The backbone (N, C alpha, C = O) RMSD from the average structure of an ensemble of 20 structures was 0.73 +/- 0.22 and 0.48 +/- 0.14 A for apoC-I(7-24) and apoC-I(35-53), respectively. In the presence of SDS, the distance geometry and simulated annealing calculations show that both peptides adopt well-defined amphipathic helices with distinct hydrophobic and hydrophilic faces. The calculated structures are discussed relative to predicted structures. Comparing our CD and NMR results for the apoC-I fragments in SDS with CD results of others obtained in the presence of dimyristoylphosphatidylcholine indicates that SDS may be a better model of the lipoprotein environment.

Effect of the Arrangement of Tandem Repeating Units of Class A Amphipathic α-Helixes on Lipid Interaction

Exchangeable apolipoproteins possess tandem repeating units of class A amphipathic helical segments and many of them are linked together by proline residues. To understand the optimal arrangement of the amphipathic helixes for lipid association, we have studied the interactions of three model class A amphipathic helical peptides with lipids. The three peptides are: 37pA, a dimer of 18A (DWLKAFYDKVAEKLKEAF) linked together by a Pro (18A-Pro-18A); 37aA, a dimer of 18A linked together by an Ala (18A-Ala-18A); and 36A, a dimer of 18A without any linker residue (18A-18A). Circular dichroism (CD) spectra showed that the peptides are predominantly alpha-helical in aqueous and lipid environments. Temperature dependent CD studies indicated that in buffer helix stability decreases in the order 36A > 37aA > 37pA; however, in the presence of dimyristoyl phosphatidylcholine (DMPC), the above order is reversed. The retention times of the peptides on a C18 reversed-phase high performance liquid chromatography column decreased in the order 36A > 37aA > 37pA, consistent with the lengths of the nonpolar faces of the alpha-helixes being in the same order; the retention time of the parent 18A was shorter than 37pA. While 37pA adsorbed to egg phosphatidylcholine monolayers most strongly, the degree and rate of association of 36A were significantly lower. Differential scanning calorimetry indicated that, while 37pA was most effective in reducing the enthalpy of the gel to liquid-crystalline phase transition of DMPC multilamellar vesicles, 36A was least effective; 36A was even less effective than 18A. Fluorescence quenching experiments with iodide and acrylamide indicated that, in the presence of DMPC, Trp residues in 36A are most exposed to the quenchers while in 37pA they are least exposed. In the presence of DMPC, shielding of Trp in 18A from the quenchers was more than that observed with Trp residues in 36A. The results of this study suggest that the arrangement of tandem repeating amphipathic helical units which results in the formation of a class A amphipathic helix with a nonpolar face longer than five or six turns reduces the ability of the helix to associate with phospholipid.

Photofrin photosensitization. Correlation of Photofrin:erythrocyte binding processes with photolysis.

Unilamellar suspensions of dimyristoylphosphatidylcholine (DMPC) can be utilized to remove Photofrin from the erythrocyte. This enables correlation of the Photofrin membrane-binding processes with Photofrin-sensitized photolysis. The observed rates of erythrocyte biding as well as the observed rates of removal of PHotofrin from the erythrocyte membrane suggest the existence of two Photofrin species that differ in their rates of exchange between the erythrocyte and buffer phases. Selective depletion and readdition of these Photofrin species to the erythrocyte membrane permits evaluation of their separate and joint photolytic efficiencies. These rapidly and slowly exchanging membrane-bound Photofrin species are separately much less efficient photosensitizers than the two species together. The two Photofrin species exhibit essentially identical fluorescence emission spectra in the presence of DMPC. Nevertheless, models consistent with the results involve partitioning by chemically distinct Photofrin components or partitioning of chemically similar Photofrin components into distinct membrane environments, or a combination of these.

Biophysical studies on fragments of the alpha-factor receptor protein.

The receptor for the alpha-factor mating pheromone of the yeast Saccharomyces cerevisiae consists of 431 amino acid residues and is a member of a family of membrane proteins predicted to have seven transmembrane helices. Fragments of the receptor corresponding to two of the transmembrane helices [residues 246-269 (M6) and 273-302 (M7)], two of the interhelical loops [residues 107-125 (E2) and 191-206 (E3)], and to a portion of the carboxyl terminus [residues 350-372 (CT)] were synthesized using solid-phase methodologies and purified to near homogeneity. CD was used to characterize the secondary structure of these peptides in trifluoroethanol (TFE), in TFE/water mixtures, in sodium dodecyl sulfate (SDS), and in the presence of dimyristoyl phosphatidylcholine (DMPC) liposomes. In TFE, M6 and M7 exhibited CD spectra consistent with highly helical peptides, whereas CT was partially helical. In contrast, E2 and E3 were either disordered or aggregated in this solvent. M6 did not partition well into DMPC vesicles whereas M7 remained helical. Both M6 and M7 assumed helical conformations in 25 mM SDS. The loop peptides and the carboxyl terminus peptide were either in a beta-structure or disordered in the presence of lipid. These findings represent the first biophysical evidence for conformations assumed by specific segments of the STE2 receptor protein.

Human Erythrocyte Band 3: Solubilization and Reconstitution into Two-dimensional Crystals

Various polyoxyethylene alkylethers were used to extract integral proteins from human erythrocyte membranes. The solubilization power of these detergents and the oligomerization of solubilized band 3 were studied. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed that short-chain detergents induced oligomers larger than the band 3 dimer. In contrast, after solubilization with long-chain detergents, the predominant band on SDS-containing gels was the monomeric band 3. Oligomerization in short-chain detergents occurred preferentially at room temperature whereas monomeric band 3 prevailed at 4°C. Consistent with these results, negative stain electron microscopy of solubilized isolated band 3 showed larger complexes with short-chain detergents than with long-chain detergents. Cu 2+/ o-phenanthroline-induced crosslinking had no effect on size or shape of band 3 particles. Despite their rather heterogeneous dimensions, octylpolyoxyethylene-solubilized band 3 complexes assembled into two-dimensional trigonal lattices ( a = b = 11(±0·5)nm) in the presence of dimyristoyl phosphatidylcholine. The unit cell exhibited a pronounced stain-filled region surrounded by three elongated morphological subunits. Each subunit most likely represents a band 3 dimer. Freeze-drying/metal-shadowing of reconstituted lattices revealed one large elevation per unit cell protruding from an otherwise smooth surface.

Architecture of Peptide Assembly in Liposome

AbstractTwo chains of amphiphilic α-helical dodecapeptides were connected to a cyclic octapeptide in a parallel or antiparallel arrangement. Conformation, orientation, and association of the dodeca-peptide chains in the presence of dimyristoylphosphatidylcholine (DMPC) liposome were studied. The peptides were easily incorporated into the bilayer membrane. CD measurements revealed that the dodecapeptides took a distorted a-helix conformation due to intramolecular association. Fluorescence quenching measurement of a probe connected to each terminal region of the dodecapeptides revealed that the dodecapeptide chain was located on the membrane surface with a parallel orientation to the surface. Thermal denaturation of the helical structure was suppressed by connecting the two dodecapeptide chains to the cyclic peptide. These experimental results indicate that a supersecondary structure composed of associated α-helices was constructed on the surface of DMPC liposome by using a cyclic peptide as a template.

Lecithin:cholesterol acyltransferase activation by synthetic amphipathic peptides.

The amphipathic helical theory of Segrest and colleagues (FEBS Lett.:38:247-253, 1974) proposes that the lipid-binding segments of serum apolipoproteins are in an alpha helical conformation. Furthermore the helices have a hydrophobic face and a hydrophilic face with a specific distribution of positively and negatively charged residues. The importance of the pattern of the charged residues in the lipid binding and lecithin:cholesterol acyltransferase (LCAT) activation by the segments is still debated. We designed a 30-residue peptide, GALA, which in the alpha helical conformation has a hydrophilic face composed of glutamic acid residues (Sabbarao et al.: Biochemistry 26:2964-2972, 1987). GALA behaves like the serum apolipoproteins in its interaction with dimyristoylphosphatidylcholine (DMPC) at neutral pH; the amino terminal tryptophan of GALA undergoes a blue shift in its fluorescence emission spectrum, and the circular dichroism (CD) spectrum indicates that GALA acquires alpha helical structure in the presence of DMPC. A DMPC-GALA:19/1 (molar ratio) complex can be isolated by gel-permeation chromatography. This complex has a discoidal structure with the approximate dimensions of 44-A edge thickness and a 170- to 350-A diameter. GALA activates LCAT with DMPC but not with unsaturated phospholipids as the substrate. The apparent partition coefficient of GALA into DMPC vesicles is 100-fold larger than into egg phosphatidylcholine vesicles. The interaction of GALA with unsaturated lipids at neutral pH is so weak that no detectable change in the spectroscopic properties of GALA or the structure of the liposomes can be detected under the conditions used here. The sequence of GALA differs from previously studied model Apo A1 peptides by the absence of positively charged residues on the hydrophilic face. This indicates that positive charges in Apo A1-like peptides are not required in order to form discoidal structures with saturated phospholipids or to activate LCAT with such lipid substrates.

Hydrolysis of stereoisomeric α-tocopheryl acetates catalyzed by bovine cholesterol esterase

The kinetics of the bovine cholesterol esterase-catalyzed hydrolysis of three stereoisomers of α-tocopheryl acetate (αT-Ac) have been examined in vitro at 37°C in the presence of dimyristoylphosphatidylcholine and sodium cholate. In contrast to in vivo results obtained earlier in rats (Ingold, K.U., Burton, G.W., Foster, D.O., Hughes, L., Lindsay, D.A. and Webb, A. (1987) Lipids 22, 163–172), 2 R,4' R,8' R- αT- Ac ( RRR- αT-Ac) is hydrolyzed (to form “natural” vitamin E) more slowly (by a factor of approx. 7) than SRR-(and SSS-αT-Ac. It is concluded that chirality at position 2 plays the dominant role in determining V max . The K n values show that RRR-αT-Ac is 2.1- and 2.7-times more strongly bound to the enzyme than are the SRR- and SSS-αT-Ac, respectively. The reaction is subject to competitive inhibition by the product with RRR-aT being 2.3-times as powerful an inhibitor as SRR--αT.

Structural analysis and amphiphilic properties of a chemically synthesized mitochondrial signal peptide.

Anionic phospholipids induce a marked conformational change in a synthetic peptide corresponding to residues 1-27 of pre-ornithine carbamyltransferase. The peptide designated, pO-(1-27)-peptide amide, becomes more alpha-helical in the presence of cardiolipin or dimyristoylphosphatidylglycerol but not in the presence of dimyristoylphosphatidylcholine. The greater helix-promoting action of anionic versus zwitterionic lipids is predicted by helix-coil transition theory. This statistical mechanical theory also predicts that a shorter peptide, N-acetyl-pO-(16-27)-peptide amide, has less helix-forming tendency, even in the presence of sodium dodecyl sulfate, despite the fact that it has a comparable number of positive charges. The N-acetyl-pO-(16-27)-peptide amide has no helical structure in buffer with or without dimyristoylphosphatidylglycerol but it has a small (5%) helical content in methanol. Thus, the ability of anionic lipids to promote helix formation requires more than the presence of cationic groups on the peptide. The angular dependence of the hydrophobic moment of the putative helical segment of pO-(1-27)-peptide amide demonstrates that any helical structure which is formed would have some amphiphilic character. The pO-(1-27)-peptide amide disrupts large lipid aggregates to form discoid micelles about 30 to 50 nm in diameter. The ability to lyse membranes into disc-shaped micelles is characteristic of peptides containing an amphiphathic helix. In the case of the mitochondrial signal peptide, this membrane-lytic behavior may contribute to the translocation of the protein into the organelle.