Structural phase transitions involved in the interaction of phospholipid bilayers with octyl glucoside

The transitional stages induced by the interaction of the nonionic surfactant octyl glucoside (OcOse) on phosphatidylcholine liposomes were studied by means of transmission electron microscopy (TEM), light scattering and permeability changes. A linear correlation was observed between the effective surfactant/lipid molar ratio (Re; three-stage model proposed for liposome solubilization) and the OcOse concentration in the initial and final interaction stages, despite showing almost a constant value during bilayer saturation. The bilayer/aqueous phase partition coefficient (K) decreased in the subsolubilizing interaction steps and increased during solubilization. Thus, whereas a preferential distribution of surfactant monomers in the aqueous phase with respect to the lipid bilayers took place in the initial interaction steps, a larger association of OcOse molecules with these lipids in bilayers occurred during solubilization. The initial steps of bilayer saturation (50-70% permeability) were attained for a lower free surfactant (Sw) than that for its critical micellar concentration (cmc). When Sw reached the OcOse cmc, solubilization started to occur (Resat). Large unilamellar vesicles began to form as the OcOse exceeded 60 mol/100 mol, exhibiting for 65 mol/100 mol (50% permeability) vesicles of approximately 400 nm. TEM pictures for 100% permeability (72 mol/100 mol) and Resat still showed unilamellar vesicles, albeit that the Resat TEM picture showing traces of smaller structures. Exceeding surfactant amounts led to a decrease in static light scattering; the vesicle-size curve began to show a bimodal distribution. The TEM picture showed tubular structures together with bilayer fragments. Thereafter, the open structures were gradually affected by the surfactant and the scattered intensity gradually decreased to a constant low value.     

Mechanism of ion transport through lipid bilayer-membranes mediated by peptide cyclo-(D-Val-L-Pro-L-Val-D-Pro)

The cyclic dodecapeptide PV, cyclo-(D-Val-L-Pro-L-Val-D-Pro)3, a structural analogue of the ion-carrier valinomycin, increases the cation permeability of lipid bilayer membranes. This paper reports the results of two types of relaxation experiments, namely relaxation of the membrane current after a voltage jump and decay of the membrane voltage after a charge pulse in lipid bilayer membranes exposed to PV. From the relaxation data, the rate constant for the translocation of the ion carrier complex across the membrane, as well as the partition coefficient of the complex between water and membrane solution interface were computed and found to be about one order of magnitude less than the comparable values for valinomycin (Val). Furthermore, the dependence of the initial membrane conductivity on ion concentration was used to evaluate the equilibrium constant, K, of complexation between PV and some monovalent cations in water. The values of K yield the following selectivity sequence of PV: Na+ less than NH4+ less than K+ less than Cs+ less than Rb+. These and earlier results are consistent with the idea that PV promotes cation movement across membranes by the solution complexation mechanism which involves complexation between ion and carrier in the aqueous phase and transport of the carrier across the membrane. In the particular form of the solution complexation mechanism operating here, the PV present in the PV-cation complex carrying charge across the membrane derives from the side from which the current is flowing (cis-mechanism). As shown previously, valinomycin, in contrast to PV, acts by an interfacial complexation mechanism in which the Val in the Val-cation complex derives from the side toward which current is flowing (trans-mechanism). The comparison of the kinetic properties of these two closely related compounds yields interesting insights into the relationship between chemical structure and function of ion carriers.     

The pH-dependence in the partitioning behaviour of (RS)-[3H]propranolol between MDCK cell lipid vesicles and buffer

PURPOSE: The pH-dependent partitioning of (RS)-[3H]propranolol between unilamellar vesicles of MDCK cell lipids and buffer was determined. METHODS: Partitioning studies were performed by means of equilibrium dialysis at 37 degrees C between pH 7 and 11 at a molar propranolol/lipid ratio in the membrane of 10(-6). RESULTS: The partition-pH diagram was bell-shaped. The highest apparent partition coefficient was 1797 at pH 9.7, the lowest was 805 at pH 6.9. Curve fitting with a combination of Henderson-Hasselbalch equations revealed an inflection point at the apparent pKa of propranolol, i.e. 9.7, and two additional pKa values at pH 7.7 and 10.0. The first one corresponds to the pKa of free fatty acids (FFA) within lipid bilayers and the other one to the pKa of phosphatidylethanolamine (PhE). The true partition coefficients (P) of the neutral as well as the ionised solute were fitted for each ionisation status of the membrane. The highest P, i.e. 2123, was calculated for neutral propranolol in the membrane with deprotonated FFA and protonated PhE. CONCLUSIONS: The partitioning behaviour of (RS)-[3H]propranolol in a complex membrane/buffer system can be described when considering ionisation changes of drug and lipids.     

Vasculopathy and insulin resistance in the JCR:LA-cp rat

The fluorescent dye FM1-43 labels nerve terminals in an activity-dependent fashion and has been found increasingly useful in exploring the exo- and endocytosis of synaptic vesicles and other cells by fluorescence methods. The dye distributes between the aqueous phase and the lipid membrane but the physical-chemical parameters characterizing the adsorption/partition equilibrium have not yet been determined. Fluorescence spectroscopy alone is not sufficient for a detailed elucidation of the adsorption mechanism since the method can be applied only in a rather narrow low-concentration window. In addition to fluorescence spectroscopy, we have therefore employed high sensitivity isothermal titration calorimetry (ITC) and deuterium magnetic resonance (2H-NMR). ITC allows the measurement of the adsorption isotherm up to 100 microM dye concentration whereas 2H-NMR provides information on the location of the dye with respect to the plane of the membrane. Dye adsorption/partition isotherms were measured for neutral and negatively-charged phospholipid vesicles. A non-linear dependence between the extent of adsorption and the free dye concentration was observed. Though the adsorption was mainly driven by the insertion of the non-polar part of the dye into the hydrophobic membrane interior, the adsorption equilibrium was further modulated by an electrostatic attraction/repulsion interaction of the cationic dye (z=+2) with the membrane surface. The Gouy-Chapman theory was employed to separate electrostatic and hydrophobic effects. After correcting for electrostatic effects, the dye-membrane interaction could be described by a simple partition equilibrium (Xb=Kcdye) with a partition constant of 103-104 M-1, a partition enthalpy of DeltaH=-2.0 kcal/mol and a free energy of binding of DeltaG=-7.8 kcal/mol. The insertion of FM1-43 into lipid membranes at room temperature is thus an entropy-driven reaction following the classical hydrophobic effect. Deuterium nuclear magnetic resonance provided insight into the structural changes of the lipid bilayer induced by the insertion of FM1-43. The dye disturbed the packing of the fatty acyl chains and decreased the fatty acyl chain order. FM1-43 also induced a conformational change in the phosphocholine headgroup. The -P-N+ dipole was parallel to the membrane surface in the absence of dye and was rotated with its positive end towards the water phase upon dye insertion. The extent of rotation was, however, much smaller than that induced by other cationic molecules of similar charge, suggesting an alignment of FM1-43 such that the POPC phosphate group is sandwiched by the two quaternary FM1-43 ammonium groups. In such an arrangement the two cationic charges counteract each other in a rotation of the -P-N+ dipole.     

Use of affinity capillary electrophoresis for the study of protein and drug interactions

Protein-drug interactions were studied using affinity capillary electrophoresis (ACE). The initial study was performed using a model system, fibronectin-heparin interaction. Two distinct binding constants, 21 and 641 nM, were derived from the Scatchard plots. The results are consistent with reported data obtained using other analytical techniques. The ACE binding assay was applied for studying molecular interactions between kedarcidin chromophore and apoprotein. Conditions with an organic solvent as buffer component were examined to establish a suitable binding assay. It appears that the electrophoretic behavior of the protein shows little distortion in the presence of either dimethyl sulfoxide (up to 10%) or acetonitrile (ACN) (up to 30%). The binding assay was initially conducted in aqueous buffer phase. The saturation concentration of chomophore was found to be around 15 microM. A linear Scatchard plot was derived from binding data with a correlation coefficient of 0.94. The binding constant was determined as Kd = 5.6 microM. The effects of organic solvent content ranging from 0 to 30% ACN on the constant were examined. The binding constants were determined as Kd = 11, 12.5 and 16.2 microM for 5, 10 and 30% ACN, respectively. It appeared that the binding affinity between kedarcidin chromophore and apoprotein is reduced as the organic solvent content in the aqueous phase is increased.     

Hydrophobicity and sorption of chlorophenolates to lipid membranes

We have studied sorption of ionized species of chlorophenols and pentahalophenols to lipid membranes using egg-phosphatidylcholine (egg-PC) vesicles and measuring their zeta-potential as a function of aqueous concentration of the phenolates. The zeta-potential isotherms can be understood in terms of a sorption model that is a combination of the Gouy-Chapman model of the electrical double layer at the membrane-water interface and the Langmuir model for sorption. Two intrinsic sorption parameters were determined: the linear partition coefficient beta m, which relates the membrane surface density of the phenolates to their aqueous concentration and the area of the adsorption site, Ps. The linear partition coefficient is the measure of the affinity of phenolates to the lipid membrane. It depends strongly on the molecular structure: 2,6-dichlorophenolate beta m = (0.45 +/- 0.08) x 10(-7); m; 3,5-dichlorophenolate beta m = (0.22 +/- 0.02) x 10(-6) m; 2,4,6-trichlorophenolate beta m = (0.63 +/-0.06) x 10(-6) m; 2,4,5-trichlorophenolate beta m = (0.11 +/- 0.01) x 10(-5) m; 2,3,5,6-tetrachlorophenolate beta m = (0.56 +/- 0.07) x 10(-5) m; 2,3,4,5-tetrachlorophenolate beta m = (0.55 +/- 0.06) x 10(-5) m; pentachlorophenolate beta m = (0.34 +/- 0.05) x 10(-4) m; pentafluorophenolate beta m = (1.00 +/- 0.13) x 10(-7) m and pentabromophenolate beta m = (0.19 +/- 0.04) x 10(-3) m. Ps was found to be independent of phenolate structure, Ps = 3.3 +/- 0.1 nm2. The membrane affinity of chlorophenolates was compared with the octanol-water partition coefficients of un-ionized chlorophenols. It was shown that the free energy of transfer of chlorophenolates from water into the lipid membrane can be divided into non-electrostatic and electrostatic contributions. The no-nelectrostatic contribution corresponds to the hydrophobicity parameter alpha = 3.94 +/- 0.0.08 kcal per nm2 of molecular surface area. The electrostatic contribution contains a term inversely proportional to the molecular radius of the phenolate ion which has the physical meaning of the work of transfer of the phenolate ion from water into the membrane. The polarity of the sorption region of egg-PC membranes is given in terms of the dielectric constant and was estimated to be 12.4 (range 10.5-13.4).     

Partition behavior of tropane alkaloids between organic solvents and water

The partition behavior of tropane alkaloids (TrA) between organic solvent and water was studied under various conditions. The apparent partition coefficient, log P', increases with pH with a slope of +1 in the acidic to neutral pH range, then tends to approach to a constant value in the alkaline region. Thus the partition of TrA to the organic phase is based on the neutral form of TrA. From the pH dependency of the partition and pH titration of TrA, the intrinsic partition coefficients log P0 and pKa values for each TrA were estimated. Values of pKa and log P0 for scopolamine were smaller than those for atropine or homatropine. It was considered that the presence of the epoxide ring in scopolamine molecule reduces the log P0 and pKa because of its polar and electro-inductive effect. It was also expected from the results of pH-dependent partition behavior that in the low pH region some portion of TrA cations is transferred to the organic phase by forming an ion-pair complex with an anion, in the aqueous phase. The log P' of TrA was measured in the presence of various kinds of anions (halides and alkyl sulfonates, C5-C8) at about pH 2, where almost all TrA are present as a protonated form. The relations between the log P' and the logarithmic concentration of anions is linear at low concentrations of anions as expected from the ion-pair partition equation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transport kinetics of hydrophobic ions in lipid bilayer membranes. Charge-pulse relaxation studies

A modified version of the charge-pulse relaxation technique with improved time resolution was applied to the study of transport kinetics of hydrophobic ions (tetraphenylborate, dipicrylamine) through lipid bilayer membranes. Besides a better time resolution the charge-pulse method has the additional advantage that the perturbation of the membrane can be kept small (voltage amplitudes between 1 and 10 mV). The results of the analysis support the model proposed earlier, according to which the overall transport takes place in three consecutive steps, adsorption of the ion from water to the interface, translocation to the opposite interface, and desorption into the aqueous phase. The translocation rate constant ki and the partition coefficient gamma of the hydrophobic ion between water and the membrane were measured for lecithins with different mono-unsaturated fatty acid residues. Increasing the chain length of the fatty acid from C16 to C24 resulted in a decrease of ki by a factor of about 9 in the case of tetraphenylborate and by a factor of about 17 in the case of dipicrylamine.     

Solvent extraction equilibria of copper and nickel with SME 529

Distribution equilibria in the extraction of copper and nickel from 1.0 mol/dm³ aqueous ammonium nitrate solution by anti-2-hydroxy-5-nonylacetophenone oxime, the active species of SME 529, in MSB 210 diluent were studied at 30°C. The extraction equilibrium constants for copper and nickel were determined to be 1.9 × 10¹ and 3.2 × 10⁵, respectively. The aqueous solubility of the oxime was also examined, resulting in a measurement of the partition coefficient of the monomeric oxime between the aqueous and organic phases, its dissociation constant in the aqueous phase and its dimerization constant in the organic phase as 2.2 × 10^?4, 0.53 nmol/dm³ and 31 dm³/mol, respectively. It was found that the extraction of copper is decreased by the addition of p-nonylphenol due to adduct formation. The equilibrium constant of the adduct formation between the oxime and p-nonylphenol was determined to be 6.6 dm³/mol. It was observed that nickel is extracted by p-nonylphenol.     

Kinetics of permeation of nucleotide through oil/water interface in the interaction of nucleotide with octadecylamine and dodecyl guanidine

The transports of tritiated ATP, ADP and AMP from the aqueous to scintillator phase with and without octadecylamine (or dodecyl guanidine) have been studied by the layered scintillation method and a theory suitable for an explanation of the results has been presented. (1) Transport processes were all expressed by the first order kinetics. (2) For the simple partitioning of ATP, the reciprocal of the rate constant of the backward permeation was linear with respect to the square of the partition coefficient. (3) For the transport of nucleotide with chemical reaction, the reciprocal of the rate constant of the backward permeation was linear against the overall partition coefficient of nucleotide. (4) A theory was presented on the basis of a general diffusion equation by assuming the two-film model with potential energy near the interface. (5) The theory could explain the dependences of the permeation rates on the partition coefficients. (6) From the finding that the ratio of the apparent diffusion coefficient in aqueous to scintillator phase was much smaller than unity, the occurrence of an energy barrier at interface was suggested. For the simple partitioning of ATP, the energy barrier was not significant.     

Transport of long-chain native fatty acids across lipid bilayer membranes indicates that transbilayer flip-flop is rate limiting

Evidence from a number of laboratories suggests that membrane proteins may meditate the transport of physiologic fatty acids (FA) across cell membranes. However, studies using lipid membranes indicate that FA are capable of spontaneous flip-flip, raising the possibility that rapid transport through the lipid phase obviates the need for a transport protein. Determining the rate-limiting steps for transport of FA across lipid membranes, therefore, is central to understanding FA transport across cell membranes. The transport of long-chain FA across lipid membranes, from the aqueous compartment on one side of the lipid bilayer to the aqueous phase on the other side, has not been measured previously. In this study, we have used the fluorescent probe ADIFAB to monitor the time course of FA movement from the outer to the inner aqueous compartments and from the lipid membrane to the outer aqueous compartment of lipid vesicles. These two measurements, together with measurements of the lipid:aqueous partition coefficients, allowed the determination of the rate constants for binding (kon), flip-flop (kff), and dissociation (koff) for the transport of long-chain natural FA across lipid vesicles. These rates were determined using large unilamellar vesicles (LUV) of approximately 1000 A diameter, prepared by extrusion and giant unilamellar vesicles (GUV), prepared by detergent dialysis, that are >/=2000 A diameter. The results of these studies for vesicles composed of egg phosphatidylcholine (EPC) and cholesterol reveal kff values that range from 3 to 15 s-1 for LUV and from 0.1 to 1.0 s-1 for GUV, depending upon temperature and FA type. For these same vesicles, dissociation rate constants range from 4 to 40 s-1 for LUV and from 0.3 to 2.5 s-1 for GUV. In all instances, the rate constant for flip-flop is smaller than koff, and because the rate of binding is greater than the rate of transport, we conclude that flip-flop is the rate-limiting step for transport. These results demonstrate that (1) kff and koff are smaller for GUV than for LUV, (2) the rate constants increase with FA type according to oleate (18:1) < palmitate (16:0) < linoleate (18:2), and (3) the barrier for flip-flop has a significant enthalpic component. Comparison of the flip-flop rates determined for GUV with values estimated from previously reported metabolic rates for cardiac myocytes, raises the possibility that flip-flop across the lipid phase alone may not be able to support metabolic requirements.     

Scanning gel chromatography: determination of transport parameters from dynamic profiles measured at low solute concentration

Direct optical scanning of solute boundaries in large zone gel chromatography experiments provides an accurate means of determining boundary profile shapes and rates of motion. A method has been developed for correcting such boundaries to a constant time frame, eliminating the distortion which arises from finite column scanning rate. Centroids of the corrected profiles can be used to determine the partition cross section for the solute of interest. The partition cross section and flow rate determine translational motion within the column. The axial dispersion coefficient, L, which characterizes rate of boundary spreading may also be calculated from the profiles. In order to explore these procedures a study of four noninteracting solutes was conducted. Partition cross sections determined from rates of motion of boundary centroids were found to be in good agreement with those determined by the equilibrium saturation method on the same column. In order to explore the lowest concentration limits of the technique and to illustrate the boundary characteristics for a self-associating solute, a study of carboxyhemoglobin was conducted over a wide concentration range. From measurements at 220 nm the lowest concentration where useful data could be obtained was 2 micrograms per ml (0.12muM heme). These results establish validity of the procedures used in analyzing the rates of boundary transport and in studying solute transport over a wide range of conditions.     

Thermodynamics of partitioning of the antimalarial drug mefloquine in phospholipid bilayers and bulk solvents

The antimalarial drug mefloquine binds avidly to phospholipids in biomembranes. The thermodynamics of the partitioning process in dimyristoylphosphatidylcholine (DMPC) bilayers was investigated to give some insight into the drug-phospholipid interaction. Thermodynamic parameters for the partition equilibria were evaluated from the equilibrium partition coefficients measured as a function of temperature. Negative values of delta H and delta S were obtained for the transfer of mefloquine from the aqueous to the gel phase of the phospholipid. The partitioning is enthalpy controlled which suggests that mefloquine interacts strongly with the phospholipid phase. In contrast, the partitioning of mefloquine into the liquid crystalline phase of DMPC is entropy controlled which is typical of a hydrophobic interaction between mefloquine and the aqueous phase. The partitioning of mefloquine into the bulk solvents octanol and hexane were found to be enthalpy and entropy controlled, respectively. The enthalpy dominated partitioning of mefloquine into gel phase DMPC and octanol is attributed to the occurrence of hydrogen bonding and van der Waals interactions between solute and solvent. The flat shape of mefloquine may further aid its interaction with the orderly domains of the lipidic/organic phase. This is apparent from a comparison of the partitioning characteristics of another structurally related but conformationally different molecule, quinine into DMPC and octanol.     

The COOH-terminal peptide binding domain is essential for self-association of the molecular chaperone HSC70

We have previously shown that the molecular chaperone HSC70 self-associates in solution into dimers, trimers, and probably high order oligomers, according to a slow temperature- and concentration-dependent equilibrium that is shifted toward the monomer upon binding of ATP peptides or unfolded proteins. To determine the structural basis of HSC70 self-association, the oligomerization properties of the isolated amino- and carboxyl-terminal domains of this protein have been analyzed by gel electrophoresis, size exclusion chromatography, and analytical ultracentrifugation. Whereas the amino-terminal ATPase domain (residues 1-384) was found to be monomeric in solution even at high concentrations, the carboxyl-terminal peptide binding domain (residues 385-646) exists as a slow temperature- and concentration-dependent equilibrium involving monomers, dimers, and trimers. The association equilibrium constant obtained for this domain alone is on the order of 10(5) M-1, very close to that determined previously for the entire protein, suggesting that self-association of HSC70 is determined solely by its carboxyl-terminal domain. Furthermore, oligomerization of the isolated carboxyl-terminal peptide binding domain is, like that of the entire protein, reversed by peptide binding, indicating that self-association of the protein may be mediated by the peptide binding site and, as such, should play a role in the regulation of HSC70 chaperone function. A general model for self-association of HSP70 is proposed in which the protein is in equilibrium between two states differing by the conformation of their carboxyl-terminal domain and their self-association properties.     

Protein partition between the different phases comprising poly(ethylene glycol)-salt aqueous two-phase systems, hydrophobic interaction chromatography and precipitation: a generic description in terms of salting-out effects

The solution behaviour of selected proteins has been studied under conditions promoting precipitation, binding to mildly hydrophobic adsorbents or partition. Solvophobic theory may be used to describe these forms of protein partition. The tendency of a protein to partition therein is dependent upon surface properties of the protein solute mediated by the concentration and nature of added salts. As applied to partitioning in poly(ethylene glycol) (PEG)-salt systems this implies that linear (Br?nsted) relationships apply only to proteins partitioned close to the critical point. At longer tie-line lengths protein partitioning is increasingly influenced by salting-out forces. This is confirmed by the observed behaviour of the proteins. The point at which this behaviour changes has been unambiguously defined enabling the direct comparison of phase transition of proteins during partition in all systems. The results obtained show that phase transition during adsorption and partition occur at similar concentrations of salt. This is less than that required to promote precipitation. It appears, from these limited studies, that top-phase preferring proteins are partitioned at salt concentrations above those required to cause adsorption. Proteins preferring the lower phase are partitioned at salt concentrations close to or below those required for adsorption. This raises questions regarding the solvated molecular form of the partitioned proteins and the definition of the partition coefficient.     

The effect of macroscopic solute diffusion in the liquid upon surface macrosegregation

An existing one-dimensional mathematical model that predicts the macrosegregation formation due to solidification shrinkage has been modified to also account for the macroscopic diffusion of solute in the liquid. It is shown both numerically and analytically that such solute diffusion has an almost negligible influence on the predicted solute profile, except in a very thin layer near the chill surface, where severe solute depletion develops. This layer is related to a discontinuity in the diffusive solute flux at the surface, and for a moderately cooled Al-4.5 pct Cu alloy, the layer thickness is of the order 100 μm. When the lever rule is imposed, the solute concentration at the surface becomes equal to the partition coefficient multiplied by the nominal alloy concentration, and the boundary layer solidifies completely once the temperature drops below the liquidus temperature of the (initial) melt. This indicates that assuming local thermodynamical equilibrium at a domain boundary when simultaneously accounting for the macroscale solute diffusion should be reconsidered in macrosegregation modeling.     

Efficient estimation of diffusion during dendritic solidification

A very efficient finite difference method has been developed to estimate the solute redistribution during solidification with diffusion in the solid. This method is validated by comparing our computed results to the results of an analytical solution derived by Kobayashi^[4] for the as-sumptions of a constant diffusion coefficient, a constant equilibrium partition ratio, and a par-abolic rate of the advancement of the solid/liquid interface. The flexibility of our method is demonstrated by applying it to the dendritic solidification of a Pb-15 wt pct Sn alloy, for which the equilibrium partition ratio and diffusion coefficient vary substantially during solidification. The fraction eutectic at the end of solidification is also obtained by estimating the fraction solid, in greater resolution, where the concentration of solute in the interdendritic liquid reaches the eutectic composition of the alloy. TURBO PASCAL is a trademark of Borland International, Scotts Valley, CA. IBM PC is a trademark of International Business Machines Corporation, Armonk, NY.     

Characterization of the oligomeric states of RecA protein: monomeric RecA protein can form a nucleoprotein filament

Self-assembly of RecA protein in solution and on single-stranded DNA exerts a significant effect on the catalytic activities of this protein. To manipulate the self-association reaction, we examined the effects of various salts on the self-association of RecA from Thermus thermophilus (ttRecA) by circular dichroism spectroscopy and gel-filtration analysis. We showed that the self-association of ttRecA strongly depends on the kind and concentration of the salt, as well as on the protein concentration. Chaotropic ions were especially useful for obtaining RecA in its hexameric and monomeric states. On the basis of these observations, we were able to regulate the oligomeric states of ttRecA and we then examined the activity of RecA in various oligomeric states. Monomeric ttRecA bound to ssDNA and formed a nucleoprotein filament, which showed ssDNA-dependent ATPase activity. These results suggest that the monomeric form of RecA is an intermediate in filament formation on ssDNA.     

Effects of liquidus and solidus curvature in solidification modeling of binary systems with constant partition ratio

A microsegregation model is used to investigate the effect of approximating liquidus and solidus lines in binary phase diagrams by straight lines during solidification modeling. Even if repartitioning of solute can be described by a constant partition coefficient, the curvature of the phase boundary lines exerts an influence on results of microsegregation calculations. Deviations of liquids and solidus lines from linearity have a distinct influence on microstructural parameters predicted for a wide range of cooling conditions, owing to the effect of pronounced changes of the solidified fraction at a given temperature. Results obtained with simplified phase diagrams should therefore be considered with care.     

Agonist-induced displacement of quinacrine from its binding site on the nicotinic acetylcholine receptor: plausible agonist membrane partitioning mechanism

It was previously demonstrated that high concentrations of cholinergic agonists such as acetylcholine (ACh), carbamylcholine (CCh), suberyldicholine (SubCh) and spin-labelled acetylcholine (SL-ACh) displaced quinacrine from its high-affinity binding site located at the lipid-protein interface of the nicotinic acetylcholine receptor (AChR) (Anas, H. R. and Johnson, D. A. (1995) Biochemistry, 34, 1589-1595). In order to account for the agonist self-inhibitory binding site which overlaps, at least partially, with the quinacrine binding site, we determined the partition coefficient (Kp) of these agonists relative to the local anaesthetic tetracaine in AChR native membranes from Torpedo californica electric organ by examining (1) the ability of tetracaine and SL-ACh to quench membrane-partitioned 1-pyrenedecanoic acid (C10-Py) monomer fluorescence, and (2) the ability of ACh, CCh and SubCh to induce an increase in the excimer/monomer ratio of C10-Py-labelled AChR membrane fluorescence. To further assess the differences in agonist accessibility to the quinacrine binding site, we calculated the agonist concentration in the lipid membrane (CM) at an external agonist concentration high enough to inhibit 50% of quinacrine binding (IC50), which in turn was obtained by agonist back titration of AChR-bound quinacrine. Initial experiments established that high agonist concentrations do not affect either transmembrane proton concentration equilibria (pH) of AChR membrane suspension or AChR-bound quinacrine fluorescence spectra. The agonist membrane partitioning experiments indicated relatively small (< or = 20) Kp values relative to tetracaine. These values follow the order: SL-ACh>SubCh>CCh-ACh. A direct correlation was observed between Kp and the apparent inhibition constant (Ki) for agonists to displace AChR-bound quinacrine. Particularly, agonist with high KpS such as SL-ACh and SubCh showed low Ki values, and this relationship was opposite for CCh and ACh. The calculated CM values indicated significant (between 7 and 54 mM) agonist accessibility to lipid membrane. By themselves, these results support the conjecture that agonist self-inhibition seems to be mediated by the quinacrine binding site via a membrane approach mechanism. The existence of an agonist self-inhibitory binding site, not located in the channel lumen would indicate an allosteric mechanism of ion channel inhibition; however, we can not discard that the process of agonist self-inhibition can also be mediated by a steric blockage of the ion channel.