Gel-liquid crystalline transition of some multilamellar lipid bilayers follows classical kinetics with a fractional dimensionality of approximately two.

The relaxation kinetics of the gel-liquid crystalline transition of phosphatidylcholine (DC14PC, DC16PC, and DC18PC) multilamellar vesicles have been examined using volume-perturbation calorimetry. The time-dependent temperature and pressure changes associated with a periodic volume perturbation are monitored in real time. Data collected in the time domain are transformed to the frequency domain using Fourier series representations of the perturbation and response functions. Because a very small perturbation is imposed during the experiment, linear response theory is suitable for analysis of the relaxation process. The Laplace transform of the classical Kolmogorov-Avrami relation of transition kinetics is used to describe the dynamic response in the frequency domain. For DC14PC and DC16PC, the relaxation process is better fit with an effective dimensionality of n = 2 rather than n = 1. For DC18PC, we estimate that an effective dimensionality of approximately 1.5 will best fit the data. These results indicate that the gel-liquid crystalline transition of these lipid bilayers follows the classical Kolmogorov-Avrami kinetic model with an effective dimensionality greater than 1 and the assumption of simple exponential decay (n = 1) commonly used in data analysis may not always be valid for lipid transitions. Insofar as the dimensionality of the relaxation reflects the geometry of fluctuating lipid clusters, this parameter may be useful in connecting experimental thermodynamic and kinetic results with those obtained from Monte Carlo simulations.     

Mechanism of local anesthetic-induced disruption of raft-like ordered membrane domains

BackgroundBecause ordered membrane domains, called lipid rafts, regulate activation of ion channels related to the nerve pulse, lipids rafts are thought to be a possible target for anesthetic molecules. To understand the mechanism of anesthetic action, we examined influence of representative local anesthetics (LAs); dibucaine, tetracaine, and lidocaine, on raft-like liquid-ordered (L_o)/non-raft-like liquid-disordered (L_d) phase separation.MethodsImpact of LAs on the phase separation was observed by fluorescent microscopy. LA-induced perturbation of the L_o and L_d membranes was examined by DPH anisotropy measurements. Incorporation of LAs to the membranes was examined by fluorescent anisotropy of LAs. The biding location of the LAs was indicated by small angle x-ray diffraction (SAXD).ResultsFluorescent experiments showed that dibucaine eliminated the phase separation the most effectively, followed by tetracaine and lidocaine. The disruption of the phase separation can be explained by their disordering effects on the L_o membrane. SAXD and other experiments further suggested that dibucaine's most potent perturbation of the L_o membrane is attributable to its deeper immersion and bulky molecular structure. Tetracaine, albeit immersed in the L_o membrane as deeply as dibucaine, less perturbs the L_o membrane probably because of its smaller bulkiness. Lidocaine hardly reaches the hydrophobic region, resulting in the weakest L_o membrane perturbation.ConclusionDibcaine perturbs the L_o membrane the most effectively, followed by tetracaine and lidocaine. This ranking correlates with their anesthetic potency.General significanceThis study suggests a possible mechanistic link between anesthetic action and perturbation of lipid rafts.     

Locations and dynamical perturbations for lipids of cationic forms of procaine, tetracaine, and dibucaine in small unilamellar phosphatidylcholine vesicles as studied by nuclear Overhauser effects in 1H nuclear magnetic resonance spectroscopy

Locations and dynamical perturbations for lipids of local anesthetics (procaine . HCl, tetracaine . HCl, and dibucaine . HCl) in sonicated egg yolk phosphatidylcholine (PC) vesicles have been studied by 1H-1H nuclear Overhauser effect (NOE) measurements. It was found that tetracaine and dibucaine bind much strongly to the neutral lipids than does procaine and that their mobilities are lowered to such an extent that spin diffusion is transmitted (i.e., omega 2 tau c2 much greater than 1). The intermolecular NOEs between drugs and PC were more effective in the case of dibucaine than with tetracaine, indicating that dibucaine binds to the lipids more strongly than tetracaine; this order agrees well with that of anesthetic potency. However, it was only tetracaine that gave any appreciable dynamical perturbation to the PC vesicles when they were monitored by the extent of transfer of the negative NOE from alpha-methylene protons to choline methyls, olefinic methines, acyl methylenes and terminal methyl protons. This finding was interpreted as being due to the differences in the locations of these drugs in small unilamellar vesicles: (1) procaine interacts with lipids very weakly at the outer surface of the vesicles; (2) tetracaine binds to the lipids both at the outer and inner halves of the bilayer, inserting its rod-like molecule in a forest of acyl chains of PC; (3) dibucaine binds tightly to the polar head-group of PC, which resides only at the outer half of the bilayer vesicles. It was concluded that the relative order of anesthetic potency within these drugs can be correlated not with the ability to affect membrane fluidity but with the ability to bind to lipids at the polar head-group of the bilayer vesicles.     

Interaction of amphotericin B with membrane lipids as viewed by 2H-NMR

The effects of amphotericin B upon the organization and dynamics of multibilayer membranes of dimyristoylphosphatidylcholine (DMPC) were investigated by means of 2H-NMR. At high amphotericin B concentrations (30 mol% with respect to the lipid) and at temperatures above 25 degrees C, DMPC experiences two different environments which are in slow exchange on the 2H-NMR time scale. In one of these, the lipid is immobilized by the antibiotic, in a molar ratio of approximately 1:1, whereas the lipid unsequestered by amphotericin B is more ordered than in its pure state. This ordering effect is perceived at relatively low antibiotic doses (4%). The local lipid order, and the relative percentage, of sequestered DMPC, are temperature-independent (up to 65 degrees C), whereas the ordering of the unsequestered lipid domain is not. The perturbation induced by amphotericin B is manifest similarly at the edges as well as in the center of the bilayer. Antibiotic addition leads to large decreases in the transverse relaxation time, T2, of the labelled lipid, but not in the spin-lattice relaxation time, T1. This indicates an increased density of slow motional modes and little change in rapid motions.     

Hydrolysis of phosphatidylcholine liposomes by phospholipases A2. Effects of the local anesthetic dibucaine.

(1) Dibucaine evokes a downward shift in the phase transition temperature of saturated phosphatidylcholines, while it also affects the pretransition. (2) The binding of dibucaine to phosphatidylcholine liposomes increases sharply when the lipid is transformed from the gel phase to the liquid-crystalline phase. (3) The activity of Naja naja phospholipase A2 towards dimyristoyl phosphatidylcholine liposomes is either stimulated or inhibited by dibucaine, depending on whether the substrate is in the gel or the liquid-crystalline state, respectively, whereas the activity of pancreatic phospholipase A2 is inhibited by the anesthetic irrespective of the physical state of the substrate. This observation is further substantiated by the results of studies on liposomes prepared from mixtures of dimyristoyl and dipalmitoyl phosphatidylcholine or dilauroyl and distearoyl phosphatidylcholine. (4) The uptake of dibucaine by positively charged liposomes composed of phosphatidylcholine and stearylamine is considerably reduced in comparison with pure phosphatidylcholine liposomes. This decrease is paralleled by a reduction of the inhibitory and stimulatory effects of dibucaine on the hydrolysis of such liposomes by pancreatic and Naja naja phospholipase, respectively. (5) The inhibitory action of dibucaine towards the pancreatic phospholipase is lowered by increasing CaCl2 concentrations. This reduction is accompanied by a decreased uptake of anesthetic by the liposomes.     

Interaction of amphotericin B with membrane lipids as viewed by H-NMR

The effects of amphotericin B upon the organization and dynamics of multibilayer membranes of dimyristoylphosphatidylcholine (DMPC) were investigated by means of ²H-NMR. At high amphotericin B concentrations (30 mol% with respect to the lipid) and at temperatures above 25°C, DMPC experiences two different environments which are in slow exchange on the ²H-NMR time scale. In one of these, the lipid is immobilized by the antibiotic, in a molar ratio of approximately 1:1, whereas the lipid unsequestered by amphotericin B is more ordered than in its pure state. This ordering effect is perceived at relatively low antibiotic doses (4%). The local lipid order, and the relative percentage, of sequestered DMPC, are temperature-independent (up to 65°C), whereas the ordering of the unsequestered lipid domain is not. The perturbation induced by amphotericin B is manifest similarly at the edges as well as in the center of the bilayer. Antibiotic addition leads to large decreases in the transverse relaxation time, T₂, of the labelled lipid, but not in the spin-lattice relaxation time, T₁. This indicates an increased density of slow motional modes and little change in rapid motions.     

Effect of the glycosphingolipid, GM1 on localization of dibucaine in phospholipid vesicles: a fluorescence study

Interaction of the local anesthetic dibucaine with small unilamellar vesicles of dimyristoylphosphatidylcholine (DMPC) and dioleoylphosphatidylcholine (DOPC) containing different mole percents of monosialoganglioside (GM1) has been studied by fluorescence spectroscopy. Fluorescence measurements on dibucaine in the presence of phospholipid vesicles containing various amounts of GM1 yielded a pattern of variation of wavelength at emission maximum and steady-state anisotropy which indicated that the microenvironment of dibucaine is more hydrophobic and rigid in membranes that contain GM1 than in membranes without it. Experiments on quenching of fluorescence from membrane-associated dibucaine by potassium iodide showed reduced quenching efficiency with the increase in GM1 content of the vesicles, demonstrating lesser accessibility of the iodide quenchers to dibucaine in the presence of GM1, when compared to that in its absence. Total emission intensity decay profiles of dibucaine yielded two lifetime components of 1 and 2.8–3.1 ns with mean relative contributions of 25 and 75%, respectively. The mean lifetime in vesicles was 20–30% lower than in the aqueous medium and showed a definite increase in presence of GM1 from that in the absence of it. All the spectral properties point that dibucaine encountered regions of membrane containing significant amount of GM1 and penetrated deeper in hydrophobic core of the bilayer.     

Peptide-induced membrane elastic deformations decelerate gramicidin dimer-monomer equilibration

Gramicidin A (gA) is a hydrophobic pentadecapeptide readily incorporating into a planar bilayer lipid membrane (BLM), thereby inducing a large macroscopic current across the BLM. This current results from ion-channel formation due to head-to-head transbilayer dimerization of gA monomers with rapidly established monomer-dimer equilibrium. Any disturbance of the equilibrium, e.g., by sensitized photoinactivation of a portion of gA monomers, causes relaxation toward a new equilibrium state. According to previous studies, the characteristic relaxation time of the gA-mediated electric current decreases as the current increases upon elevating the gA concentration in the membrane. Here, we report data on the current relaxation kinetics for gA analogs with N-terminal valine replaced by glycine or tyrosine. Surprisingly, the relaxation time increased rather than decreased upon elevation of the total membrane conductance induced by these gA analogs, thus contradicting the classical kinetic scheme. We developed a general theoretical model that accounts for lateral interaction of monomers and dimers mediated by membrane elastic deformations. The modified kinetic scheme of the gramicidin dimerization predicts the reverse dependence of the relaxation time on membrane conductance for gA analogs, with a decreased dimerization constant that is in a good agreement with our experimental data. The equilibration process may be also modulated by incorporation of other peptides (“impurities”) into the lipid membrane.     

Fractional order viscoelasticity of the aortic valve cusp: an alternative to quasilinear viscoelasticity

BACKGROUND: Quasilinear viscoelasticity (QLV) theory has been widely and successfully used to describe the time-dependent response of connective tissues. Difficulties remain, however, particularly in material parameter estimation and sensitivities. In this study, we introduce a new alternative: the fractional order viscoelasticity (FOV) theory, which uses a fractional order integral to describe the relaxation response. FOV implies a fractal-like tissue structure, reflecting the hierarchical arrangement of collagenous tissues. METHOD OF APPROACH: A one-dimensional (I-D) FOV reduced relaxation function was developed, replacing the QLV "box-spectrum" function with a fractional relaxation function. A direct-fit, global optimization method was used to estimate material parameters from stress relaxation tests on aortic valve tissue. RESULTS: We found that for the aortic heart valve, FOV had similar accuracy and better parameter sensitivity than QLV, particularly for the long time constant (tau2). The mean (n = 5) fractional order was 0.29, indicating that the viscoelastic response of the tissue was strongly fractal-like. RESULTS SUMMARY: mean QLV parameters were C = 0.079, tau1 = 0.004, tau2 = 76, and mean FOV parameters were beta = 0.29, tau = 0.076, and rho = 1.84. CONCLUSIONS: FOV can provide valuable new insights into tissue viscoelastic behavior Determining the fractional order can provide a new and sensitive quantitative measure for tissue comparison.     

Local anesthetics and pressure: a comparison of dibucaine binding to lipid monolayers and bilayers

The binding of the local anesthetic dibucaine to monolayers composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine was studied with a Langmuir trough at pH 5.5 (22 degrees C, 0.1 M NaCl). At this pH value only the charged form of the local anesthetic exists in solution. Charged dibucaine was found to be surface active and to penetrate into the lipid monolayer, with the hydrophobic part of the molecule being accommodated between the fatty acyl chains of the lipid. The dibucaine intercalation could be quantitated by measuring the expansion of the film area, delta A, at constant surface pressure, pi. At a given surface pressure, delta A increased with increasing dibucaine in the buffer phase. On the other hand, keeping the dibucaine concentration constant, the area increase, delta A, was strongly dependent on the surface pressure. The area increase, delta A, was large at low surface pressure and decreased with increasing surface pressure. A plot of the relative change in surface area, delta A/A, versus the surface pressure yielded straight lines in the pressure range of 25-36 mN/m for five different concentrations. The delta A/A vs. pi isotherms intersected at pi = 39.5 +/- 1 mN/m with delta A = O, indicating that charged dibucaine apparently can no longer penetrate into the monolayer film. By making judicial assumptions about the area requirement of dibucaine the monolayer expansion curves could be transformed into true binding isotherms. Dibucaine binding isotherms were constructed for different monolayer pressures and were compared to a bilayer binding isotherm measured under similar conditions with ultraviolet spectroscopy. The best agreement between monolayer and bilayer binding data was obtained for a monolayer held at a pressure of 30.7 to 32.5 mN/m, which can thus be considered as the bilayer-monolayer equivalence pressure. It is further suggested from this analogy that the binding of dibucaine does not change the internal pressure in the bilayer phase, at least not in the concentration range of physiological interest (0-2 mM dibucaine) but induces a lateral expansion. At higher molar ratios of cationic dibucaine to lipid, chi b, in the monolayer (chi b greater than 0.20) the area increase is larger than would be expected from the molecular dimensions of dibucaine. This is probably due to charge repulsion effects, which at still higher molar ratios (chi b greater than 0.6) lead to a micellisation. The pressure dependence of the intercalation of cationic dibucaine into lipid membranes may also be of relevance for the phenomenon of pressure reversal in anesthesia.     

Lipid-protein interactions of the human erythrocyte concanavalin a receptor in phospholipid bilayers

The interaction of the human erythrocyte concanavalin A receptor (a subpopulation of Band 3) with phospholipids has been investigated using differential scanning microcalorimetry of reconstituted vesicles prepared by detergent dialysis. The mean diameter of dialyzed phospholipid vesicles jumps dramatically on inclusion of the concanavalin A receptor and then increases linearly with the fraction of protein in the bilayer. The glycoprotein has a dramatic effect on the phospholipid gel to liquid-crystalline phase transition, and ΔH decreases linearly with increasing mole fraction of protein up to a protein/lipid mole ratio of around 1:1160. Extrapolation of this data indicates that each concanavalin A receptor is able to perturb about 685 molecules of dimyristoylphosphatidylcholine, withdrawing them from the main phase transition. The cooperativity of phospholipid melting is profoundly disrupted by small amounts of glycoprotein, with the cooperative unit dropping to less than half its initial values at a protein/lipid mole ratio of 1:3800. A break occurs in the ΔH curve as the protein/lipid mole ratio is increased above 1:1160, and ΔH then increases linearly with increasing amounts of concanavalin A receptor in the bilayer. This phenomenon may be interpreted in terms of protein-protein aggregation which occurs in the phospholipid bilayer above a certain critical mole fraction of concanavalin A receptor, resulting in perturbed phospholipids being returned to the phase transition. In addition, the hydrophilic domains of the glycoprotein may exist in two different conformations depending on the protein concentration in the bilayer, and these may differ in their ability to interact with phospholipid headgroups at the membrane surface.     

Stimulation and inhibition of pancreatic phospholipase A2 by local anesthetics as a result of their interaction with the substrate.

1. At low concentrations the local anesthetic dibucaine stimulates hydrolysis by pancreatic phospholipase A2 of phospholipids extracted from rat liver mitochondria or microsomes, whereas at higher concentrations it inhibits. The action of this enzyme towards membrane-bound substrates is barely influenced by low, but inhibited by high concentrations of dibucaine. 2. Butacaine, which is a weaker anesthetic, stimulates hydrolysis of extracted phospholipids and inhibits that of membrane-bound substrates, both actions being concentration dependent. 3. The inhibitory potency of dibucaine is several times higher in NaCl than in sucrose solutions and strongly increases with decreasing pH. Neither one of these two effects is the result of a change in binding efficiency of the anesthetic to the substrates. 4. Extracted total membrane lipids bind considerably less anesthetic than an equivalent amount of native membrane. Liver phosphatidylethanolamine is more effective in binding of dibucaine than liver phosphatidylcholine. 5. Binding of dibucaine to the phospholipase, as studied by equilibrium dialysis is at the lower level of detectability. According to the same method dibucaine is unable to displace 45Ca2+ bound to the enzyme. 6. These results are interpreted as to support the view that local anesthetics interfere with pancreatic phospholipase activity by means of interaction with the substrate rather than with the enzyme.     

A comparative study of the effects of procaine, lidocaine, tetracaine and dibucaine on the functions and ultrastructure of isolated rat liver mitochondria

The effects of procaine, lidocaine, tetracaine and dibucaine (10(-5) - 10(-2) M) were tested on isolated rat liver mitochondria by measurements of the respiratory rates and of the membrane potential and by electron microscopy. A general concentration-dependent stimulation of the basal state (respiration before ADP addition) was observed for all local anesthetics studied. Up to the concentration of 10(-3) M, the order of stimulation was: procaine less than lidocaine less than dibucaine less than tetracaine. However, with the exception of dibucaine, which inhibited state-3 respiration (ADP present) in a strictly concentration-dependent manner, the other drugs had a biphasic effect: slight stimulation of state 3 at low and moderate concentrations (less than or equal to 10(-3) M) and inhibition at higher concentrations. Nevertheless, due to a stronger stimulation of the basal state, the acceptor control ratio decreases progressively (uncoupling effect) as the concentration of the drugs increases. The only exception to this observation is procaine in the range of 10(-5) - 10(-4) M, where the stimulation of the two respiration states (although small) is approximately equal and thus the uncoupling effect is absent or negligible. Membrane potential recordings suggested that membrane integrity and phosphorylation capacity were negatively affected at high drug concentrations (greater than 10(-3) M), especially in the case of tetracaine and dibucaine, when 5 x 10(-3) M even produced the collapse of the membrane potential and complete loss of the phosphorylation ability. Electron microscopy confirmed these effects, showing an abundance of either swollen or supercondensed mitochondria, with many membrane ruptures. The action mechanisms of the tertiary amines studied are discussed in terms of interaction of drug with the lipid bilayer and with the membrane proteins. It is concluded that both the inhibitory and the uncoupling effects are dependent, in the first place, on the degree of hydrophobicity of each local anesthetic.     

Dynamics of drug-DNA interactions: a comparative temperature jump study of ellipticinium and 9-hydroxy ellipticinium

The temperature-jump method has been used to compare the binding of 2-N methyl ellipticinium (NME) and 2-N methyl 9 hydroxy ellipticinium (NMHE) to three natural DNA's of different AT/GC composition. The relaxation signals, analyzed by the Padé-Laplace method, are characterized by two distinct relaxation times, tau 1 and tau 2, respectively in the 1-4 ms and 20-80 ms range. In the case of the NMHE/DNA interaction, the slower relaxation time tau 2 depends on the DNA composition, as follows: tau 2 (Micrococcus lysodeikticus) greater than tau 2 (Calf thymus) greater than tau 2 (Clostridium perfringens). Contrary to NMHE, NME which does not possess an OH group at the C-9 position, shows no relaxation time dependence upon DNA base composition. The observation of two relaxation times indicates that the binding equilibria are associated with at least two distinct drug/DNA complexes (probably arising from two distinct DNA binding sites). Three kinetic models, involving the formation of a weak intermediate ionic complex, are given to explain the binding reaction between these cationic drugs and the DNA. They allow the determination of the four rate constants associated with the two binding steps and lead to equilibrium association constants in agreement with those obtained from spectroscopic studies. The validity of the models is discussed and it is shown that the best kinetic scheme, for either NMHE or NME, could be that in which the ionic step is not a prerequiste to intercalation. The kinetic results show that the residence time of 9 hydroxy ellipticinium is markedly increased in GC rich DNA's and this could be related to the higher in vitro and in vivo cytotoxic properties of 9 hydroxy substituted ellipticines.     

Effects of melittin on molecular dynamics and Ca-ATPase activity in sarcoplasmic reticulum membranes: electron paramagnetic resonance.

We have performed electron paramagnetic resonance (EPR) experiments on nitroxide spin labels incorporated into rabbit skeletal sarcoplasmic reticulum (SR), in order to investigate the physical and functional interactions between melittin, a small basic membrane-binding peptide, and the Ca-ATPase of SR. Melittin binding to SR substantially inhibits Ca(2+)-dependent ATPase activity at 25 degrees C, with half-maximal inhibition at 9 mol of melittin bound per mole of Ca-ATPase. Saturation transfer EPR (ST-EPR) of maleimide spin-labeled Ca-ATPase showed that melittin decreases the submillisecond rotational mobility of the enzyme, with a 4-fold increase in the effective rotational correlation time (tau r) at a melittin/Ca-ATPase mole ratio of 10:1. This decreased rotational motion is consistent with melittin-induced aggregation of the Ca-ATPase. Conventional EPR was used to measure the submicrosecond rotational dynamics of spin-labeled stearic acid probes incorporated into SR. Melittin binding to SR at a melittin/Ca-ATPase mole ratio of 10:1 decreases lipid hydrocarbon chain mobility (fluidity) 25% near the surface of the membrane, but only 5% near the center of the bilayer. This gradient effect of melittin on SR fluidity suggests that melittin interacts primarily with the membrane surface. For all of these melittin effects (on enzymatic activity, protein mobility, and fluidity), increasing the ionic strength lessened the effect of melittin but did not alleviate it entirely. This is consistent with a melittin-SR interaction characterized by both hydrophobic and electrostatic forces. Since the effect of melittin on lipid fluidity alone is too small to account for the large inhibition of Ca-ATPase rotational mobility and enzymatic activity, we propose that melittin inhibits the ATPase primarily through its capacity to aggregate the enzyme, consistent with previous observations of decreased Ca-ATPase activity under conditions that decrease protein rotational mobility.     

Local anesthetics and divalent cations have the same effect on the headgroups of phosphatidylcholine and phosphatidylethanolamine

The effects of the local anesthetic dibucaine on the membrane headgroup conformations of phosphatidylcholine and phosphatidylethanolamine were determined using ²H- and ³¹P-NMR. The size of the deuterium quadrupole splittings of the two methylene segments of the choline and ethanolamine groups changed dramatically and the 31-phosphorus chemical shift anisotropy of the phosphatidylcholine headgroup decreased by about 7 ppm in the presence of local anesthetic. The quadrupole splittings of the 3-glycerol and choline methyl segments were relatively insensitive to the addition of dibucaine. The headgroup data for dibucaine addition paralleled similar data for the addition of various cations. These NMR results agree with the previous observation that these drugs displace calcium from phospholipids. The effects of this local anesthetic on these headgroups were distinctly different from the changes induced by cholesterol, heat and the general anesthetic chloroform.     

Kinetics of acetylcholine-activated cation channel blockade by the calcium antagonist D-600 inAplysia neurons

The effects of the calcium channel blocker D-600 on the cation channels activated by acetylcholine (ACh) was studied in voltage-clamped Aplysia neurons by voltage-jump relaxation analysis. D-600 blocked the steady-state ACh current in a highly voltage-dependent manner, the degree of antagonism increasing with membrane hyperpolarization. In the presence of D-600 the current relaxations following hyperpolarizing command steps became biphasic. The time constants of ACh-induced current relaxations (tau f), which approximate the mean channel lifetime, were reduced in a voltage-dependent manner, the degree of reduction of tau f increasing with increasing membrane potential. In addition to the acceleration of tau f, a slow, inverse kinetic component (tau s) of the relaxation appeared in the presence of D-600. The rate of this inverse kinetic component was accelerated either by increasing the agonist or antagonist dose or by increasing the membrane potential. These results suggest that D-600 acts to antagonize the acetylcholine response through a blockade of the open state of the transmitter-activated cation channel. Possible kinetic schemes for this interaction are discussed.     

Locations of local anesthetic dibucaine in model membranes and the interaction between dibucaine and a Na+ channel inactivation gate peptide as studied by 2H- and 1H-NMR spectroscopies.

To study the molecular mechanisms of local anesthesia, locations of local anesthetic dibucaine in model membranes and the interactions of dibucaine with a Na+ channel inactivation gate peptide have been studied by 2H- and 1H-NMR spectroscopies. The 2H-NMR spectra of dibucaine-d9 and dibucaine-d1, which are deuterated at the butoxy group and at the 3 position in its quinoline ring, respectively, have been observed in multilamellar dispersions of the lipid mixture composed of phosphatidylcholine, phosphatidylserine, and phosphatidylethanolamine. 2H-NMR spectra of deuterated palmitic acids incorporated, as a probe, into the lipid mixture containing cholesterol have also been observed. An order parameter, SCD, for each carbon segment was calculated from the observed quadrupole splittings. Combining these results, we concluded that first, the butoxy group of dibucaine is penetrating between the acyl chains of lipids in the model membranes, and second, the quinoline ring of dibucaine is located at the polar region of lipids but not at the hydrophobic acyl chain moiety. These results mean that dibucaine is situated in a favorable position that permits it to interact with a cluster of hydrophobic amino acids (Ile-Phe-Met) within the intracellular linker between domains III and IV of Na+ channel protein, which functions as an inactivation gate. To confirm whether the dibucaine molecule at the surface region of lipids can really interact with the hydrophobic amino acids, we synthesized a model peptide that includes the hydrophobic amino acids (Ac-GGQDIFMTEEQK-OH, MP-1), the amino acid sequence of which corresponds to the linker part of rat brain type IIA Na+ channel, and the one in which Phe has been substituted by Gln (MP-2), and measured 1H-NMR spectra in both phosphate buffer and phosphatidylserine liposomes. It was found that the quinoline ring of dibucaine can interact with the aromatic ring of Phe by stacking of the rings; moreover, the interaction can be reinforced by the presence of lipids. In conclusion, we wish to propose that local anesthesia originates from the pi-stacking interaction between aromatic rings of an anesthetic molecule located at the polar headgroup region of the so-called boundary lipids and of the Phe in the intracellular linker between domains III and IV of the Na+ channel protein, prolonging the inactivated state and consequently making it impossible to proceed to the resting state.     

Kinetic studies on the helix-coil transition of fluorescent labeled poly(-L-lysine) by the temperature-jump technique

Fluorescent dansyl labels were covalently attached to poly (L-lysine) (poly(Lys)) with a degree of polymerization of 300 to 600. The degree of labeling was 0.01 to 0.085 (mol label to mol amino acid residues). From the decay of the anisotropy of fluorescence it was concluded that the labels were highly mobile both in the coiled and helical state. A decrease of fluorescence intensity accompanied the helix-coil transition. Identical pH induced transition curves were measured by circular dichroism and fluorescence. The midpoint of the transition was at pH 10.2. The kinetics of the transition were studied by temperature-jump relaxation using fluorescence detection. A single relaxation phase was observed. The relaxation time tau exhibited a distorted bell shaped dependence on the degree of helicity f with a maximum value tau(max) = 15 micros at f = 0.3 and 20 degrees C. It was independent of polymer concentration and of the degree of labeling. A rate constant of helix propagation kF = 10(7) s(-1) was calculated from tau(max) and published values of the nucleation parameter sigma. The activation energy was 16 kJ mol . The observed rate constant is comparable to that of poly(L-glutamic acid) but two orders of magnitude smaller than that found for polyamino acids with nonionizable side chains.     

Kinetics of gelatin transitions with phase separation: T-jump and step-wise DSC study

The isothermal gelation (or melting) of gelatin after fast cooling (or heating) steps is studied by using high sensitivity differential scanning micro-calorimetry, in order to determine the dependence of the kinetic and thermodynamic parameters upon changes in composition and in temperature. The calorimetric heat flow curves, obtained according to defined temperature profiles, have been fitted with exponential functions (simple exponentials or stretched exponentials for the step-wise and for T-jump experiments, respectively). The gelation process of gelatin alone for t<300 min shows that the characteristic time tau and the fractional exponent are beta very sensitive to the concentration of gelatin chains and to the microscopic phase segregation due to the presence of another polymeric component.