Paclitaxel partitioning into lipid bilayers

Sequence variation in the src gene product could, in principle, influence metastasis formation through either of 2 effects: an alteration in tumor antigenicity or a non-immune-mediated change in one or more src-associated functions. Our present results establish that both mechanisms underlie the difference in relative levels of metastasis formation induced by the v-src vs. the c-src(527) oncogene. A point that emerges from this analysis is the segregation, within a chicken line genotypically uniform at the major histocompatibility (B) complex (MHC), of a phenotype defined by strong resistance to secondary v-src-induced tumor challenge. The pattern of segregation is consonant with the possibility that a gene unlinked to the MHC governs immune response levels to v-src-encoded tumor antigen.     

Structural studies of polymer-cushioned lipid bilayers

The structure of softly supported polymer-cushioned lipid bilayers, prepared in two different ways at the quartz-solution interface, were determined using neutron reflectometry. The polymer cushion consisted of a thin layer of branched, cationic polyethyleneimine (PEI), and the bilayers were formed by adsorption of small unilamellar dimyristoylphosphatidylcholine (DMPC) vesicles. When vesicles were first allowed to adsorb to a bare quartz substrate, an almost perfect bilayer formed. When the polymer was then added to the aqueous solution, it appeared to diffuse beneath this bilayer, effectively lifting it from the substrate. In contrast, if the polymer layer is adsorbed first to the bare quartz substrate followed by addition of vesicles to the solution, there is very little interaction of the vesicles with the polymer layer, and the result is a complex structure most likely consisting of patchy multilayers or adsorbed vesicles.     

Hydrophobic interactions of peptides with membrane interfaces

Intestinal glycohydrolases are enzymes involved in assimilating carbohydrate for nutrition. The avian forms of these enzymes, in particular the maltase-glucoamylase complex (MG), are not well characterised. This study encompassed characterisation of these enzymes from ostrich intestines, and the first kinetic analysis of an avian MG. Proteolytically solubilised MG from ileal brush border membrane vesicles was purified by Sephadex G-200 gel filtration and Tris-affinity-chromatography, while jejunal sucrase-isomaltase (SI) and MG were purified by Toyopearl-Q650 and phenyl-Sepharose chromatography. Amino acid sequences and compositions of enzyme subunits, resulting from SDS-PAGE, were determined. Kinetics of hydrolysis of linear oligosaccharides was studied. Ostrich MG and SI showed the highest activity in the jejunum, followed by the ileum and duodenum. No lactase or trehalase activity could be detected. The jejunal MG and SI, resulting from brush-border membrane vesicles, could not be separated during purification. However, a minor form of ileal MG was purified using Sephadex G-200 chromatography. Ileal MG contained three subunits of M(r) 145,000, 125,000 and 115,000. Although the N-terminal amino acid sequences bear no homology to SI, the M(r) 115,000 subunit shows homology to porcine MG in both sequence and amino acid composition. The pH optimum of maltose-, starch- and isomaltose-hydrolysing activity was 6.5 and that of sucrose-hydrolysing activity 5.5. The glycohydrolases were most active at 58 degrees C, but were quickly denatured above 60 degrees C. Sucrose- and starch-hydrolysing activities were more thermostable than maltose- and isomaltose-hydrolysing activities. Kinetic parameters (K(m), kcat and kcat/K(m)) for the hydrolysis of maltooligosaccharides, starch and glycogen are reported for ileal MG. Maltotriose and maltotetraose displayed partial inhibition of ileal MG. The study revealed large similarities between ostrich SI and MG in charge, size, shape and hydrophobicity, based on their inseparability by several methods. Measurement of the specificity constants for maltooligosaccharide hydrolysis by ileal MG revealed less efficient hydrolysis of longer substrates as compared to maltose and maltotriose.     

Lateral diffusion in planar lipid bilayers

Direct measurements by fluorescence correlation spectroscopy of lateral diffusion coefficients of fluorescent lipid analogs in lipid bilaryer membranes indicate self-diffusion coefficients D greater than 10(-7) square centimeters per second for various lipid systems above their reported transition temperatures. Cholesterol in egg lecithin at mole ratio of 1 : 2 reduces D by about twofold, while retained hydrocarbon solvent can increase it by two- to threefold.     

Mechanism of alamethicin insertion into lipid bilayers

Alamethicin adsorbs on the membrane surface at low peptide concentrations. However, above a critical peptide-to-lipid ratio (P/L), a fraction of the peptide molecules insert in the membrane. This critical ratio is lipid dependent. For diphytanoyl phosphatidylcholine it is about 1/40. At even higher concentrations P/L > or = 1/15, all of the alamethicin inserts into the membrane and forms well-defined pores as detected by neutron in-plane scattering. A previous x-ray diffraction measurement showed that alamethicin adsorbed on the surface has the effect of thinning the bilayer in proportion to the peptide concentration. A theoretical study showed that the energy cost of membrane thinning can indeed lead to peptide insertion. This paper extends the previous studies to the high-concentration region P/L > 1/40. X-ray diffraction shows that the bilayer thickness increases with the peptide concentration for P/L > 1/23 as the insertion approaches 100%. The thickness change with the percentage of insertion is consistent with the assumption that the hydrocarbon region of the bilayer matches the hydrophobic region of the inserted peptide. The elastic energy of a lipid bilayer including both adsorption and insertion of peptide is discussed. The Gibbs free energy is calculated as a function of P/L and the percentage of insertion phi in a simplified one-dimensional model. The model exhibits an insertion phase transition in qualitative agreement with the data. We conclude that the membrane deformation energy is the major driving force for the alamethicin insertion transition.     

Theory of self-assembly of lipid bilayers and vesicles

A simple theory is developed that explains the formation of bilayers and vesicles and accounts quantitatively for many of their physical properties: Properties including vesicle size distributions and bilayer elasticity emerge from a unified theory that links thermodynamics, interaction free energy, and molecular geometry. The theory may be applied to the analysis of more complicated membrane structures and mechanisms.     

Molecular organization and dynamics of 1-palmitoyl-2-oleoylphosphatidylcholine bilayers containing a transmembrane alpha-helical peptide

The molecular organization and dynamics have been investigated in membranes consisting of 1-palmitoyl-2-oleoyl-l-alpha-phosphatidylcholine (POPC) and various ratios of a transmembrane alpha-helical peptide, Ac-K2L24K2-amide (L24), in order to gain insights into how the transmembrane portions of membrane proteins are mixed with phospholipids and organized in biological membranes. Particular attention was paid to membranes with high peptide concentrations. The molecular organization and dynamics were studied in the ps-to-micros regime using various spin-labeling techniques. Conventional ESR spectra as well as saturation-recovery curves measured in both the presence and the absence of molecular oxygen showed that phosphatidylcholine spin-labels detect the existence of a single homogeneous environment, indicating that both L24 and POPC are likely to be undergoing fast translational diffusion in L24-POPC membranes of up to 9 mol % peptide. Since 16-18 molecules of phosphatidylcholine are required to surround a transmembrane alpha-helical peptide [Morrow, M. R., Huschilt, J. C., and Davis, J. H. (1985) Biochemistry 24, 5396-5406], L24 must form L24-rich regions at a P/L ratio of 1/10 instantaneously. However, these results suggest that the lipid exchange rates among the bulk, boundary, and L24-rich regions are fast, and that the L24-rich regions must be forming and dispersing rapidly in a time scale shorter than 0.1 micros, the conventional ESR spin-label time scale and the electron spin-lattice relaxation time scale in the presence of molecular oxygen. Although this does not exclude the possibility of the formation of small, stable oligomers of L24, it is unlikely because L24 lacks features that would favor their formation. L24 (9 mol %) increases the hydrophobicity of the central part of the POPC membrane from the level of 1-decanol to that of pure hexane and also increases the hydrophobicity near the membrane surface from the level of 2-propanol to that of 1-decanol. The effect of 9 mol % L24 on the order parameter profile is similar to that of decreasing the temperature by approximately 8 degrees C between 10 and 55 degrees C. It is concluded that L24 is highly miscible in POPC membranes even at high concentrations in the membrane.     

Anticonvulsant activity and interactions with neuronal voltage-dependent sodium channel of analogues of ameltolide

Fifteen compounds related to ameltolide (LY 201116) were studied for (i) anticonvulsant potential in the maximal electroshock-induced seizures (MES) and the subcutaneous pentylenetetrazol (sc Ptz) tests in mice and rats and (ii) interactions with neuronal voltage-dependent sodium channels. Compounds were chosen ranging in anticonvulsant activity in mice from very active to inactive. The active compounds were defined as those protecting 50% of the animals at doses between 10 and 50 micromol/kg and inactive compounds as those protecting 50% of the animals at doses greater than 1 mmol/kg. The series studied included three N-(2,6-dimethylphenyl)benzamides (compounds 1, 2 (ameltolide), and 3), three N-(2,2,6, 6-tetramethyl)piperidinyl-4-benzamides (compounds 4, 5, 6), one phenylthiourea (compound 7), five N-(2,6-dimethylphenyl)phthalimides (compounds 8, 9, 10, 13, and 14), two N-phenylphthalimide derivatives (compounds 11 and 12), and one N-(2,2,6, 6-tetramethyl)piperidinyl-4-phthalimide (compound 15). Phenytoin (PHT) was employed as the reference prototype antiepileptic drug. After inital screening in mice, compounds 1, 2, 3, 5, 8, 9, 10, 13, and 14 were selected for further testing in rats. Anticonvulsant ED50s (effective doses in at least 50% of animals tested) of compounds in the MES test were determined in rats dosed orally and amounted to 52 (1), 135 (2), 284 (3), 231 (8), 131 (9), 25 (10), 369 (13), 354 (14), and 121 (PHT) micromol/kg, compound 5 presenting with an ED50 value higher than 650 micromol/kg. In our hands, the apparent IC50s (inhibitory concentrations 50) of compounds toward binding to rat brain synaptosomes of [3H]batrachotoxinin-A-20alpha-benzoate were 0.25 (1), 0.97 (2), 0.35 (3), 25.8 (5), 161.3 (8), 183.5 (9), 0.11 (10), 1.86 (13), 47.8 (14), and 0.86 (PHT) microM. The relationship between the activity in the MES test and the capacity to interact in vitro with neuronal voltage-dependent sodium channels and the fact that the IC50 values obtained in the in vitro test are close to the brain concentrations at which anticonvulsant activities are reported to occur for ameltolide strongly suggest that the anticonvulsant properties of most compounds tested could be a direct result of their interaction with the neuronal voltage-dependent sodium channel.     

A microscopic model for lipid/protein bilayers with critical mixing

A statistical mechanical lattice model is proposed to describe the phase diagram of phospholipid bilayers with small transmembrane proteins or polypeptides. The model is based on the extended Pink-Green-Chapman model (Zhang et al. (1992) Phys. Rev. A 45, 7560-7567) for pure lipid bilayers which undergo a first-order gel-fluid phase transition. The interaction between the lipid bilayer and the protein or polypeptide is modelled using the concept of hydrophobic matching. The phase diagram has been derived by computer-simulation techniques which fully account for thermal density fluctuations and which operate on the level of the free-energy thereby permitting an accurate identification of the phase boundaries. The calculations predict a closed loop of gel-fluid coexistence with a lower critical mixing point. Specific-heat traces across the phase diagram are also presented. The theoretical results for the phase diagram, the specific-heat function, and the transition enthalpy are related to recent experimental measurements on phospholipid bilayers mixed with synthetic transmembrane amphiphilic polypeptides or with gramicidin A.     

Structure and interactions of fully hydrated dioleoylphosphatidylcholine bilayers

This study focuses on dioleoylphosphatidylcholine (DOPC) bilayers near full hydration. Volumetric data and high-resolution synchrotron x-ray data are used in a method that compares DOPC with well determined gel phase dipalmitoylphosphatidylcholine (DPPC). The key structural quantity obtained is fully hydrated area/lipid A0 = 72.2 +/- 1.1 A2 at 30 degrees C, from which other quantities such as thickness of the bilayer are obtained. Data for samples over osmotic pressures from 0 to 56 atmospheres give an estimate for the area compressibility of KA = 188 dyn/cm. Obtaining the continuous scattering transform and electron density profiles requires correction for liquid crystal fluctuations. Quantitation of these fluctuations opens an experimental window on the fluctuation pressure, the primary repulsive interaction near full hydration. The fluctuation pressure decays exponentially with water spacing, in agreement with analytical results for soft confinement. However, the ratio of decay length lambda(fl) = 5.8 A to hydration pressure decay length lambda = 2.2 A is significantly larger than the value of 2 predicted by analytical theory and close to the ratio obtained in recent simulations. We also obtain the traditional osmotic pressure versus water spacing data. Our analysis of these data shows that estimates of the Hamaker parameter H and the bending modulus Kc are strongly coupled.     

N-palmitoyl sphingomyelin bilayers: structure and interactions with cholesterol and dipalmitoylphosphatidylcholine

The structure and thermotropic properties of N-palmitoyl sphingomyelin (C16:0-SM) and its interaction with cholesterol and dipalmitoylphosphatidylcholine (DPPC) have been studied by differential scanning calorimetry (DSC) and X-ray diffraction methods. DSC of hydrated multi-bilayers of C16:0-SM shows reversible chain-melting transitions. On heating, anhydrous C16:0-SM exhibits an endothermic transition at 75 degrees C (delta H = 4.0 kcal/mol). Increasing hydration progressively lowers the transition temperature (TM) and increases the transition enthalpy (delta H), until limiting values (TM = 41 degrees C, delta H = 7.5 kcal/mol) are observed for hydration values > 25 wt % H2O. X-ray diffraction at temperatures below (29 degrees C) TM show a bilayer gel structure (d = 73.5 A, sharp 4.2 A reflection) for C16:0-SM at full hydration; above TM, at 55 degrees C, a bilayer liquid-crystal phase is present (d = 66.6 A, diffuse 4.6 A reflection). Addition of cholesterol to C16:0-SM bilayers results in a progressive decrease in the enthalpy of the transition at 41 degrees C, and no cooperative transition is detected at > 50 mol % cholesterol. X-ray diffraction shows no difference in the bilayer periodicity, position/width of the wide-angle reflections, or electron density profiles at 29 and 55 degrees C when 50 mol % cholesterol is present. Thus, cholesterol inserts into C16:0-SM bilayers progressively removing the chain-melting transition and changing the structural characteristics of the bilayer. DSC and X-ray diffraction data show that DPPC is completely miscible with C16:0-SM bilayers in both the gel and liquid-crystalline phases; however, 30 mol % C16:0-SM removes the pre-transition exhibited by DPPC.     

Fluorescence quenching by metal ions in lipid bilayers

Fluorescence quenching of perylene by Co2+ and Ni2+ ions has been investigated both below and above the phase transition temperature in small unilamellar DPPC vesicles. Classic F?rster type energy transfer was observed for perylene quenched by Co2+ ions below the phase transition when the effects of donor-donor energy transfer are taken into account. In the liquid crystalline phase a simple diffusion theory incorporating energy transfer was found to model the system well. For quenching by Ni2+ ions both above and below the phase transition temperature in lipid bilayers and in glycerol the data did not follow classic F?rster type energy transfer but indicated that an additional quenching mechanism was present. A mechanism other than F?rster behaviour was also observed for the quenching by Cr3+ ions in glycerol.     

Multiple states of beta-sheet peptide protegrin in lipid bilayers

Protegrin-1 (PG-1), a beta-sheet antimicrobial peptide, was studied in aligned lipid bilayers by oriented circular dichroism (OCD). All of its spectra measured in a variety of lipid compositions were linear superpositions of two primary basis spectra, indicating that PG-1 existed in two different states in membranes. We designated these as state S and state I. The state assumed by PG-1 was strongly influenced by lipid composition, peptide concentration, and hydration condition. We have previously reported that the helical peptides, alamethicin and magainin, also exhibit two distinct OCD basis spectra-one corresponding to surface adsorption with the helix parallel to the bilayer and the other with perpendicular transbilayer insertion. States S and I of PG-1 may correspond to the surface state and the insertion state of alamethicin, since they show a similar dependence on lipid composition, peptide concentration, and hydration condition. Nonoriented CD spectra obtained from vesicle, micelle, and solution preparations are not linear superpositions of the basis spectra of the states S and I. This indicates that a molecular orientation change alone is insufficient to describe the S left and right arrow I transition. Rather, a more complicated process is taking place, perhaps involving a change in the hydrogen bonding pattern of the backbone. Although the structural basis of the OCD spectra remains to be determined, the discovery of two distinct states can provide information about dynamic changes of PG-1 in membranelike environments, properties undoubtedly related to its antimicrobial and cytotoxic effects.