Partition of a fluorescent molecule between liquid-crystalline and crystalline regions of membranes

Summary We have determined the partition coefficient of the fluorescent molecule perylene between liquid crystalline and crystalline regions of vesicle membranes formed from binary mixtures of several lipids. We measured the fluorescence intensity of perylene in these vesicles as a function of temperature and used the intensity profiles, together with a theory developed in a previous paper, to determine the partition coefficient defined as the ratio of the concentration of perylene in the liquid-crystalline (fluid) regions of the membrane to the concentration in the crystalline (solid) phase. In vesicles composed of dipalmitoyl phosphatidylcholine/distearoyl phosphatidylcholine (dppc/dspc) mixtures and of dipalmitoyl phosphatidylcholine/dipalmitoyl phosphatidylethanolamine (dppc/dppe) mixtures, the partition coefficient is close to unity. Its value is 1.04±0.18 for dppc/dsp mixtures and 1.10±0.26 for dppc/dppe mixtures. In vesicles composed of dimyristoyl phosphatidylcholine/distearoyl phosphatidylcholine mixtures, the partition coefficient was more difficult to determine and its value ranged from 0.3 to 7.     

Proton NMR bandshape studies of lamellar liquid crystals and gel phases containing lecithins and cholesterol.

Proton NMR spectra for gel and liquid crystalline samples, composed of dimyristoyl and/or dipalmitoyl lecithin, cholesterol and water, can be consistently interpreted in terms of mesophase symmetry and molecular diffusion according to a model proposed by Wennerstrom (Wennerstrom, H. (1973) Chem. Phys. Lett. 18, 41-44). It is shown by computer simulation that the characteristic "super-lorentzian" bandshape of the lamellar mesophase can be described by the superposition of three gaussian curves. The NMR signal of the gel phase can be simulated by the superposition of two gaussian curves with widths at half height of 2.5 kHz and 19 kHz. An upper limit of the lateral diffusion coefficient of the lecithin molecules in the gel phase is calculated to be about 5-10(-15) m-2/s. It is therefore concluded that the static intermolecular dipolar couplings average to zero in the lamellar mesophase. An estimation of the order parameter of the liquid crystalline phase is made from experimental data and a calculated "rigid lattice" linewidth. A two phase system is shown to exist in the temperature range 28-34 degrees C for a mesophase of a mixture of dimyristoyl and dipalmitoyl lecithin. The presence of cholesterol results in enhanced lateral diffusion of the lecithin molecules at temperatures below the Chapman transition point.     

Chemically induced lipid phase separation in model membranes containing charged lipids: a spin label study.

The lipid distribution in binary mixed membranes containing charged and uncharged lipids and the effect of Ca2+ and polylysine on the lipid organization was studied by the spin label technique. Dipalmitoyl phosphatidic acid was the charged, and spin labelled dipalmitoyl lecithin was the uncharged (zwitterionic) component. The ESR spectra were analyzed in terms of the spin exchange frequency, Wex. By measuring Wex as a function of the molar percentage of labelled lecithin a distinction between a random and a heterogeneous lipid distribution could be made. It is established that mixed lecithin-phosphatidic acid membranes exhibit lipid segregation (or a miscibility gap) in the fluid state. Comparative experiments with bilayer and monolayer membranes strongly suggest a lateral lipid segregation. At low lecithin concentration, aggregates containing between 25% and 40% lecithin are formed in the fluid phosphatidic acid membrane. This phase separation in membranes containing charged lipids is understandable on the basis of the Gouy-Chapman theory of electric double layers. In dipalmitoyl lecithin and in dimyristoyl phosphatidylethanolamine membranes the labelled lecithin is randomly distributed above the phase transition and has a coefficient of lateral diffusion of D = 2.8-10(-8) cm2/s at 59 degrees C. Addition of Ca2+ dramatically increases the extent of phase separation in lecithin-phosphatidic acid membranes. This chemically (and isothermally) induced phase separation is caused by the formation of crystalline patches of the Ca2+-bound phosphatidic acid. Lecithin is squeezed out from these patches of rigid lipid. The observed dependence of Wex on the Ca2+ concentration could be interpreted quantitatively on the basis of a two-cluster model. At low lecithin and Ca2+ concentration clusters containing about 30 mol % lecithin are formed. At high lecithin or Ca2+ concentrations a second type of precipitation containing 100% lecithin starts to form in addition. A one-to-one binding of divalent ions and phosphatidic acid at pH 9 was assumed. Such a one-to-one binding at pH 9 was established for the case of Mn2+ using ESR spectroscopy. Polylysine leads to the same strong increase in the lecithin segregation as Ca2+. The transition of the phosphatidic acid bound by the polypeptide is shifted from Tt = 47.5 degrees to Tt = 62 degrees C. This finding suggests the possibility of cooperative conformational changes in the lipid matrix and in the surface proteins in biological membranes.     

Membrane composition modulates thiopental partitioning in bilayers and biomembranes

The nonspecific interaction of thiopental with erythrocyte ghosts, synaptic membranes, microsomes and mitochondria has been measured at 25°C and pH 6.6. In cholesterol-depleted erythrocyte ghosts the partition coefficient decreases with increasing cholesterol content. In sonicated liposomes made from egg lecithin and cholesterol the partition coefficient also decreases with increasing cholesterol content. The dependence of the partition coefficient on cholesterol content in the biological membranes, on average, parallels that in the lipid bilayers. The partition coefficient in lipid bilayers made from lipids extracted from erythrocyte ghosts was comparable to that in the corresponding egg lecithin/cholesterol bilayer. The partition coefficients of all the biomembranes are consistently lower than those in the corresponding egg lecithin/cholesterol bilayer, the free energy of transfer between biomembrane and corresponding bilayer being ?1 kcal/mol.     

Mobility in the mitochondrial electron transport chain

The role of lateral diffusion in mitochondrial electron transport has been investigated by measuring the diffusion coefficients for lipid, cytochrome c, and cytochrome oxidase in membranes of giant mitoplasts from cuprizone-fed mice using the technique of fluorescence redistribution after photobleaching (FRAP). The diffusion coefficient of the phospholipid analogue N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine is dependent on the technique used to remove the outer mitochondrial membrane. A sonication technique yields mitoplasts with monophasic recovery of the lipid probe (D = 6 X 10(-9) cm2/s), while digitonin-treated mitochondria show biphasic recoveries (D1 = 5 X 10(-9) cm2/s; D2 = 1 X 10(-9) cm2/s). Digitonin appears to incorporate into mitoplasts, giving rise to decreased lipid mobility concomitant with increased rates of electron transfer from succinate to oxygen, in a manner reminiscent of the effects of cholesterol incorporation [Schneider, H., Lemasters, J. J., Hochli, M., & Hackenbrock, C. R. (1980) J. Biol. Chem. 255, 3748-3756]. FRAP measurements on tetramethylrhodamine cytochrome c modified at lysine-39 and on a mixture of active morpholinorhodamine derivatives of cytochrome c gave diffusion coefficients of (3.5-7) X 10(-10) cm2/s depending on the assay medium. With morpholinorhodamine-labeled antibodies purified on a cytochrome oxidase affinity column, the diffusion coefficient for cytochrome oxidase was determined to be 1.5 X 10(-10) cm2/s. The results are discussed in terms of a dynamic aggregate model in which an equilibrium exists between freely diffusing and associated electron-transfer components.     

Effect of the gel to liquid crystalline phase transition on the osmotic behaviour of phosphatidylcholine liposomes.

Aspects of osmotic properties of liposomes, prepared from synthetic lecithin, above, at and below the gel to liquid crystalline phase transition temperature are described. The experiments show that liposomal membranes with their lipids in the gel state are still permeable to water. The rate of water permeation changes drastically on passing the transition temperature. The water permeation has activation energies of 9.5 +/- 1.28 and 26.4 +/- 0.9 kcal/mol above and below the transition temperature, respectively, indicating that the diffusion processes take place by different mechanisms. With respect to the barrier properties of the liposomes in the vicinity of the transition temperature, the following conclusions can be made. (1) Studying the osmotic shrinkage of liposomes at a fixed temperature near the transition point, the experiments indicate that dimyristoyl phosphatidylcholine membranes are highly permeable to glucose under these conditions, where liquid and solid domains co-exist. Under the same conditions the osmotic experiments did not indicate a strong increase in glucose permeability of dipalmitoyl phosphatidylcholine membranes as compared to the situation above and below the transition temperature. (2) On the other hand, perturbations of the phase equilibrium by temperature varations resulted in a marked increase of the glucose permeation through dipalmitoyl phosphatidylcholine bilayers. Once a new phase equilibrium of liquid and solid regions is established the permeation rate of glucose is much less.     

Chemically induced lipid phase separation in model membranes containing charged lipids: A spin label study

The lipid distribution in binary mixed membranes containing charged and uncharged lipids and the effect of Ca²⁺ and polylysine on the lipid organization was studied by the spin label technique. Dipalmitoyl phosphatidic acid was the charged, and spin labelled dipalmitoyl lecithin was the uncharged (zwitterionic) component. The ESR spectra were analyzed in terms of the spin exchange frequency, W_ex. By measuring W_ex as a function of the molar percentage of labelled lecithin a distinction between a random and a heterogeneous lipid distribution could be made. It is established that mixed lecithinphosphatidic acid membranes exhibit lipid segregation (or a miscibility gap) in the fluid state. Comparative experiments with bilayer and monolayer membranes strongly suggest a lateral lipid segregation. At low lecithin concentration, aggregates containing between 25% and 40% lecithin are formed in the fluid phosphatidic acid membrane. This phase separation in membranes containing charged lipids is understandable on the basis of the Gouy-Chapman theory of electric double layers.In dipalmitoyl lecithin and in dimyristoyl phosphatidylethanolamine membranes the labelled lecithin is randomly distributed above the phase transition and has a coefficient of lateral diffusion of D = 2.8·10^?8 cm²/s at 59°C.Addition of Ca²⁺ dramatically increases the extent of phase separation in lecithin-phosphatidic acid membranes. This chemically (and isothermally) induced phase separation is caused by the formation of crystalline patches of the Ca²⁺-bound phosphatidic acid. Lecithin is squeezed out from these patches of rigid lipid. The observed dependence of W_ex on the Ca²⁺ concentration could be interpreted quantitatively on the basis of a two-cluster model. At low lecithin and Ca²⁺ concentration clusters containing about 30 mol% lecithin are formed. At high lecithin or Ca²⁺ concentrations a second type of precipitation containing 100% lecithin starts to form in addition. A one-to-one binding of divalent ions and phosphatidic acid at pH 9 was assumed. Such a one-to-one binding at pH 9 was established for the case of Mn²⁺ using ESR spectroscopy.Polylysine leads to the same strong increase in the lecithin segregation as Ca²⁺. The transition of the phosphatidic acid bound by the polypeptide is shifted from T_t = 47.5° to T_t = 62°C. This finding suggests the possibility of cooperative conformational changes in the lipid matrix and in the surface proteins in biological membranes.     

Surface diffusion in human serum lipoproteins

From the viscosity dependence of the 31P NMR signals, the diffusion coefficients DT of phospholipid molecules in the surface monolayer of HDL, LDL and VLDL have been determined. DT for HDL3 and HDL2 are found to be 2.3 X 10(-8) cm2/s and 1.8 X 10(-8) cm2/s, respectively. These values are similar to values reported for diffusion of phospholipid molecules in phospholipid bilayers above the gel to liquid crystalline phase transition temperature. Viscosity dependence of [16,16,16-2H3]phosphatidylcholine incorporated into HDL2 yielded a value similar to that determined by 31P (DT = 1.9 X 10(-8) cm2/s). Slower diffusion coefficients were measured for LDL2 and VLDL. VLDL had a value DT = 9.1 X 10(-9) cm2/s. The diffusion coefficient for LDL2 was 1.4 X 10(-9) cm2/s. Thus, diffusion of phospholipids in LDL2 is a full order of magnitude slower at 25 degrees C than diffusion of phospholipids in the HDLs.     

Lateral diffusion of epidermal growth factor complexed to its surface receptors does not account for the thermal sensitivity of patch formation and endocytosis

The patching and endocytosis of EGF (epidermal growth factor) bound to A-431 cells (a human epidermoid carcinoma line) are temperature-sensitive processes which are completely inhibited at 4 degrees C. Receptor-mediated endocytosis generally occurs through coated regions, and EGF bound to its membrane receptor must diffuse laterally to these points of internalization. In this work we investigated the thermal sensitivity of the lateral diffusion of EGF receptor complexes and the thermal sensitivity of the patching and endocytosis of the hormone receptor complexes. Using the fluorescence photobleach recovery technique, we measured the lateral diffusion coefficients of a fluorescent derivative of EGF as a function of temperature. The lateral diffusion coefficient (D) increased gradually from 2.8 X 10(-10) cm2/s at 5 degrees C to 8.5 X 10(-10) cm2/s at 37 degrees C, and no phase transition was detected. Neither was a phase transition detected when we measured the diffusion coefficient of fluorescent lipid probes over this temperature range. From a calculation of the collision frequency of the occupied EGF receptors with coated regions using our measured values of D at 5 and 37 degrees C, we conclude that diffusion is not the rate-limiting step for either endocytosis or patching.     

Rapid diffusion of spectrin bound to a lipid surface.

Human erythrocyte spectrin was labelled with the probe 5, 5'-disulfato-1-(6-hexanoic acid N-hydroxysuccinimide ester)-1'-ethyl-3,3,3',3'-tetramethylindocarbocyanine (Cy3). Cy3-spectrin was bound to the outer surface of dimyristoylphosphatidylcholine (DMPC) multilamellar vesicles and its diffusion measured by fluorescence recovery after photobleaching (FRAP). It was found that at 30 degrees C, above the lipid gel to liquid-crystalline phase transition of the lipids, Cy3-spectrin had an unexpectedly high diffusion coefficient D=(2.1+/-0.6)x10(-7)) cm2/s. At the phase transition, diffusion of Cy3-spectrin was only slightly lower; D=(1.3+/-0.3)x10(-7) cm2/s, whereas at 14 degrees C, well below the lipid phase transition, diffusion was found to be much slower with D=(3.1+/-0.12)x10(-9) cm2/s. The fast diffusion of Cy3-spectrin on the lipid surface implies that the individual bonds which bind spectrin to the lipid surface must rapidly be made and broken. In the light of these results, spectrin-lipid interactions alone appear unlikely to have any significant role in supporting the cell membrane. Probably, the interactions serve only to localise the spectrin at the inner lipid surface in order to facilitate formation of the cytoskeleton.     

Polymyxin binding to charged lipid membranes. An example of cooperative lipid-protein interaction.

The binding of polymyxin-B to lipid bilayer vesicles of synthetic phosphatidic acid was studied using fluorescence, ESR spectroscopy and electron microscopy. 1,6-Diphenylhexatriene (which exhibits polarized fluorescence) and pyrene decanoic acid (which forms excimers) were used as fluorescence probes to study the lipid phase transition. The polymyxin binds strongly to negatively charged lipid layers. As a result of lipid/polymyxin chain-chain interactions, the transition temperature of the lipid. This can be explained in terms of a slight expansion of the crystalline lipid lattice (Lindeman's rule). Upon addition of polymyxin to phosphatidic acid vesicles two rather sharp phase transitions (width deltaT = 5 degrees C) are observed. The upper transition (at Tu) is that of the pure lipid and the lower transition (at T1) concerns the lipid bound to the peptide. The sharpness of these transitions strongly indicates that the bilayer is characterized by a heterogeneous lateral distribution of free and bound lipid regions, one in the crystalline and the other in the fluid state. Such a domain structure was directly observed by electron microscopy (freeze etching technique). In (1 : 1) mixtures of dipalmitoyl phosphatidic acid and egg lecithin, polymyxin induces the formation of domains of charged lipid within the fluid regions of egg lecithin. With both fluorescence methods the fraction of lipid bound to polymyxin-B as a function of the peptide concentration was determined. S-shaped binding curves were obtained. The same type of binding curve is obtained for the interaction of Ca2+ with phosphatidic acid lamellae, while the binding of polylysine to such membranes is characterized by a linear or Langmuir type binding curve. The S-shaped binding curve can be explained in terms of a cooperative lipid-ligand (Ca2+, polymyxin) interaction. A model is proposed which explains the association of polymyxin within the membrane plane in terms of elastic forces caused by the elastic distortion of the (liquid crystalline) lipid layer by this highly asymmetric peptide.     

Fluorescence decay of pyrene in small and large unilamellar L,α-Dipalmitoylphosphatidylcholine vesicles above and below the phase transition temperature

The fluorescence decays of pyrene in small and large unilamellar L,-dipalmitoylphosphatidylcholine vesicles have been investigated as a function of probe concentration and temperature. When the molar ratio of pyrene to phospholipid equals 1:3000, no excimer emission is observed and the fluorescence decays are mono-exponential. When this ratio is equal to or higher than 1:120, excimer formation is observed.Above the phase transition temperature the observed fluorescence decays of monomer and excimer can be adequately described by a bi-exponential function. The monomer decays can be equally well fitted to a decay law which takes into account a time-dependence in the probe diffusion rate constant. The fluorescence decay kinetics are compatible with the excimer formation scheme which is valid in an isotropic medium. The excimer lifetime and the (apparent) rate constant of excimer formation have been determined as a function of probe concentration at different temperatures above the phase transition temperature. The activation energy of excimer formation is found to be 29.4±1.3 kJ/mol. In small unilamellar vesicles the diffusion constant associated with the pyrene excimer formation process varies from 8.0x10^-7 cm²/s at 40°C to 2.2x10^-6 cm²/s at 70°C.Below the phase transition temperature the monomer decays can be described by a decay law which takes into account a time dependence of the rate constant of excimer formation. The lateral diffusion coefficient of pyrene calculated from the decay fitting parameters of the monomer region varies from 4.0x10^-9 cm²/s at 20°C to 7.9x10^-8 cm²/s at 35°C. No significant difference could be observed between the pyrene fluorescence decay kinetics in small and large unilamellar vesicles.Abbreviations SUV small unilamellar vesicles - LUV large unilamellar vesicles - DPPC dipalmitoylphosphatidylcholine - DMPC dimyristoylphosphatidylcholine - FRAP fluorescence recovery after photobleaching Part of this research has been presented at the 5th international symposium on surfactants in solution. Bordeaux, July 9th–13th 1984     

Measurement and interpretation of fluorescence polarisations in phospholipid dispersions

An instrument that measures the temperature dependence of fluorescence polarisation and intensity directly and continuously is described. The behaviour of four fluorescent probes bound to a number of well characterised model systems was then examined. The motional properties of the probes were determined from the polarisation and intensity data and were found to be sensitive to the crystallineliquid crystalline phase transitions in phospholipid vesicles of dimyristoyl and dipalmitoyl phosphatidylcholine. Binary mixture of dilauroyl and dipalmitoyl phosphatidycholine show lateral phase separation and in this system the probes partition preferentially into the more ‘fluid’ phase. In systems that have been reported to contain ‘short range order’ or ‘liquid clustering’, such as dioleoyl phosphatidylcholine and liquid paraffin, the motion of the probes was found to have anomalous Arrhenius behaviour consistent with the idea that homogeneous phases were not being sampled. The significance of these findings for the interpretation of the behaviour of fluorescent probes bound to natural membranes is discussed.     

A method for estimating lateral diffusion coefficients in membranes from steady-state fluorescence quenching studies.

The Stern-Volmer theory, in which the quantum yield ratio (Io/I) depends linearly on the quencher concentration, will typically be inapplicable to fluorescence quenching in membranes. Numerical analysis shows that diffusion-controlled quenching results in a nonlinear concentration dependence for diffusion coefficients less than or of the order of 10(-6) cm2 s-1 and probe fluorescence lifetimes in the region of 10-100 ns. Lateral diffusion coefficients in membranes are typically overestimated an order of magnitude or more by the Stern-Volmer theory. An alternative empirical method is presented, which represents nonlinear concentration curves by a single parameter linear approximation determined by a least-squares analysis. The fitting parameter, P, depends on the interaction distance, the membrane thickness, the maximum extent of quenching and, in the case of biexponential probe fluorescence decay, the fluorescence kinetic parameters. P is presented in tabular form for a useful range of these parameters. The method is used to estimate diffusion coefficients for plastoquinone and plastoquinol from pyrene fluorescence quenching in soya bean phosphatidylcholine liposomes. It is found that the diffusion coefficients are nearly equal and in the region of 1.3-3.5 X 10(-7) cm2 s-1 for interaction radii of 1.5-0.5 nm, respectively.     

The rippled structure in bilayer membranes of phosphatidylcholine and binary mixtures of phosphatidylcholine and cholesterol

Freeze-fracture electron microscopy is used to study the rippled texture in pure dimyristoyl and dipalmitoyl phosphatidylcholine membranes and in mixtures of dimyristoyl phosphatidylcholine and cholesterol. Evidence is presented that the apparent phase transition properties of multilamellar liposomes may be dependent on the manner in which liposomes are prepared. Under certain conditions the ripple structures as visualized by freeze-fracture electron microscopy for the pure phosphatidylcholines are observed to be temperature dependent in the vicinity of the pretransition. Thus the transition can sometimes appear to be a gradual transition rather than a sharp, first-order phase transition. In mixtures of dimyristoyl phosphatidylcholine and cholesterol, the ripple repeat distance is found to increase as the cholesterol concentration is increased between 0 and 20 mol%. Above 20 mol%, no rippling is observed. A simple theory is presented for the dependence of ripple repeat spacing on cholesterol concentration in the range 0–20 mol%. This theory accounts for the otherwise inexplicable abrupt increase in the lateral diffusion coefficients of fluorescent lipids in binary mixtures of phosphatidylcholine and cholesterol when the cholesterol concentration is increased above 20 mol%.     

Phospholipid lateral organization in synthetic membranes as monitored by pyrene-labeled phospholipids: effects of temperature and prothrombin fragment 1 binding

Pyrene-labeled phospholipids have been used to test for the existence of lateral domains due to temperature-induced phase separations and binding of prothrombin fragment 1 to charged lipid vesicles. When in close proximity, pyrene-containing probes can exchange excited-state energy to form excimers; the ratio of the excimer to monomer fluorescence intensity (E/M) is proportional to the local concentration of probe in the membranes, as well as to the excimer lifetime and the probe's lateral diffusion coefficient. The ability of the pyrene-labeled phospholipids to quantitatively report the coexistence of multiple environments was demonstrated in dipalmitoylphosphatidylcholine/palmitoyloleoylphosphatidylcholine multilamellar vesicle preparations of varying compositions, each of which contained coexisting fluid and gel phases. In this system, pyrene-labeled phosphatidylcholine was found to favor the fluid relative to the gel phase with a partition coefficient of 7. At 37 degrees C, in dioleoylphosphatidylglycerol (DOPG)/palmitoyloleoylphosphatidylcholine (POPC) large, unilamellar vesicles containing either pyrene-labeled phosphatidylglycerol (py-PG) or pyrene-labeled phosphatidylcholine (py-PC), the excimer lifetime (37 ns) and the lateral diffusion constant of the probe (5.8 X 10(-8) cm2/s) were independent of the membrane composition and the presence of fragment 1 and Ca2+. Consequently, E/M was directly proportional to only the local concentration of the py-PG or py-PC probes. When saturating amounts of fragment 1 and 5 mM Ca2+ were added to DOPG/POPC vesicles that contained either probe, no change in E/M and hence the local probe concentration was observed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetic investigations on the phase transition of phospholipid bilayers

Pressure-jump experiments were performed on vesicles and liposomes of dimyristoyl phosphatidylcholine and dipalmitoyl phosphatidylcholine following the time course of solution turbidity. For both lipids two relaxation effects were evaluated the time constants of which exhibit clear maxima at the midpoint of the phase transition. The time constants lie for vesicles in the 100 μs and 1 ms ranges and for liposomes in the 1 ms and 10 ms ranges. The processes are slightly faster for dimyristoyl phosphatidylcholine than for dipalmitoyl phosphatidylcholine. All relaxation times are concentration-independent. The time constant and amplitude behaviours indicate that all processes are cooperative in agreement with previous interpretations. It is demonstrated that cooperative units can be evaluated from the relaxation amplitudes. These are of the same order of magnitude as those obtained from static experiments. On the grounds of the present kinetic investigation we can state that the application of the linear Ising model to two-dimensional processes as attempted for the static lipid phase transition is inadequate.     

Tetracaine-membrane interactions: effects of lipid composition and phase on drug partitioning, location, and ionization

Interactions of the local anesthetic tetracaine with unilamellar vesicles made of dimyristoyl or dipalmitoyl phosphatidylcholine (DMPC or DPPC), the latter without or with cholesterol, were examined by following changes in the drug's fluorescent properties. Tetracaine's location within the membrane (as indicated by the equivalent dielectric constant around the aromatic fluorophore), its membrane:buffer partition coefficients for protonated and base forms, and its apparent pK(a) when adsorbed to the membrane were determined by measuring, respectively, the saturating blue shifts of fluorescence emission at high lipid:tetracaine, the corresponding increases in fluorescence intensity at this lower wavelength with increasing lipid, and the dependence of fluorescence intensity of membrane-bound tetracaine (TTC) on solution pH. Results show that partition coefficients were greater for liquid-crystalline than solid-gel phase membranes, whether the phase was set by temperature or lipid composition, and were decreased by cholesterol; neutral TTC partitioned into membranes more strongly than the protonated species (TTCH(+)). Tetracaine's location in the membrane placed the drug's tertiary amine near the phosphate of the headgroup, its ester bond in the region of the lipids' ester bonds, and associated dipole field and the aromatic moiety near fatty acyl carbons 2-5; importantly, this location was unaffected by cholesterol and was the same for neutral and protonated tetracaine, showing that the dipole-dipole and hydrophobic interactions are the critical determinants of tetracaine's location. Tetracaine's effective pK(a) was reduced by 0.3-0.4 pH units from the solution pK(a) upon adsorption to these neutral bilayers, regardless of physical state or composition. We propose that the partitioning of tetracaine into solid-gel membranes is determined primarily by its steric accommodation between lipids, whereas in the liquid-crystalline membrane, in which the distance between lipid molecules is larger and steric hindrance is less important, hydrophobic and ionic interactions between tetracaine and lipid molecules predominate.     

Translational diffusion of bovine prothrombin fragment 1 weakly bound to supported planar membranes: measurement by total internal reflection with fluorescence pattern photobleaching recovery.

Previous work has shown that bovine prothrombin fragment 1 binds to substrate-supported planar membranes composed of phosphatidylcholine (PC) and phosphatidylserine (PS) in a Ca(2+)-specific manner. The apparent equilibrium dissociation constant is 1-15 microM, and the average membrane residency time is approximately 0.25 s-1. In the present work, fluorescence pattern photobleaching recovery with evanescent interference patterns (TIR-FPPR) has been used to measure the translational diffusion coefficients of the weakly bound fragment 1. The results show that the translational diffusion coefficients on fluid-like PS/PC planar membranes are on the order of 10(-9) cm2/s and are reduced when the fragment 1 surface density is increased. Control measurements were carried out for fragment 1 on solid-like PS/PC planar membranes. The dissociation kinetics were similar to those on fluid-like membranes, but protein translational mobility was not detected. TIR-FPPR was also used to measure the diffusion coefficient of the fluorescent lipid NBD-PC in fluid-like PS/PC planar membranes. In these measurements, the diffusion coefficient was approximately 10(-8) cm2/s, which is consistent with that measured by conventional fluorescence pattern photobleaching recovery. This work represents the first measurement of a translational diffusion coefficient for a protein weakly bound to a membrane surface.     

Diffusion, patching, and capping of stearoylated dextrans on 3T3 cell plasma membranes

Fluorescence-labeled trinitrophenylated stearoylated dextrans have been used as controllable analogues of cell membrane proteins on model membranes and on a variety of natural cell membranes. This paper reports their behavior on 3T3 mouse fibroblast plasma membranes. Spatial distribution on the membrane was studied by fluorescence microscopy, and molecular mobility was measured by fluorescence photobleaching recovery. At concentrations from 10(2) to 3 X 10(3) molecules/micron2 essentially homogeneous fluorescence was observed after treatment with these stearoyldextrans in culture. Diffusion coefficients and fractional recovery of fluorescence after photobleaching were cvoncentration independent. For 3 X 10(3) molecules/micron2 we found at 23 degrees C D = (3.0 +/- 1.8) X 10(-10) cm2/s with 65 +/- 17% recovery and at 37 degrees C D = (7.0 +/- 5.0) X 10(-10) cm2/s without a change of the fractional recovery. Cross-linking with antibodies stopped diffusion on a macroscopic scale and sometimes induced patching, mottling (defined as the development of gaps in the fluorescence layer), and capping (defined as the confinement of the fluorescence to less than 50% of the cell). Capping required approximately 3 h at 37 degrees C and was inhibited by metabolic poisons and cytochalasin B. These drugs did not affect stearoyldextran diffusion or fractional recovery. Colchicine, which did not dramatically affect capping, slowed diffusion two- to threefold but did not affect fractional recovery. The antibody inhibition of the diffusion of stearoyldextrans precedent to capping did not affect the diffusion of a lipid probe or fluorescein isothiocyanate labeled membrane proteins. When the trinitrophenylated stearoyldextran was cleared from most of the surface by capping and the surface subsequently relabeled with stearoyldextran, the diffusion coefficient and fractional recovery of the second label were identical with those of the first label prior to capping. Thus, capping does not clear an immobilizing factor from the membrane.