Absorption of surfactants by membranes: erythrocytes versus synthetic vesicles.

Three surfactants (chlorpromazine hydrochloride, thioridazine hydrochloride, and sodium deoxycholate) are found to absorb just as strongly into the protein-containing membranes of erythrocytes as into the phospholipid bilayers of synthetic vesicles. In the concentration region where hemolysis occurs and the Langmuir adsorption isotherm is no longer valid, one may use a phase partition model in which the erythrocyte membrane is one of the phases. The partition coefficients, expressed as the ratio of mole fraction surfactant in the membrane lipid phase to concentration of surfactant in the aqueous phase, have been calculated at the point of saturation in the erythrocyte membrane. These values are Ky = 430 M-1 (chlorpromazine, pH 5.9), 550 M-1 (deoxycholate, pH 7.6), and 640 M-1 (thioridazine, pH 5.9), in isotonic buffer at 27 degrees C. Corresponding values for synthetic vesicles made from dimyristoylphosphatidylcholine are Kx = 230 M-1 (chlorpromazine, 0.12 M buffer/KCl pH 5.9), 440 M-1 (deoxycholate, 0.20 M buffer/NaCl pH 8.0) and 510 M-1 (thioridazine, 0.12 M buffer/KCl pH 5.9), at 27 degrees C. It appears that the surfactants become an integral part of the bilayer in both vesicles and natural membranes and that the absorption is not of a peripheral nature. There is no evidence that the presence of proteins in the natural membrane inhibits the absorption of these surfactants in any way.     

Bovine brain phosphatidylinositol transfer protein. Selective inhibition by chlorpromazine and other amphiphilic amines

Cationic amphiphilic amines of varied pharmacological activity were evaluated as modulators of the protein-catalyzed, intermembrane transfers of phosphatidylinositol and phosphatidylcholine. The catalytic agent was brain phosphatidylinositol transfer protein; the membrane system consisted of two populations of single bilayer phospholipid vesicles. The majority of the amines tested caused decreases in phospholipid transfer activity with the relative potencies in the following order: chlorpromazine greater than dibucaine greater than propranolol much greater than tripelennamine approximately chloroquine greater than dipyridamole. Concentrations required for 50% inhibition of phosphatidylinositol transfer were 0.24 mM chlorpromazine, 0.46 mM dibucaine, and 0.78 mM propranolol. The phosphatidylcholine transfer activity of this protein was somewhat less sensitive to these compounds. Comparison of chlorpromazine and its quaternary amine analogue, methochlorpromazine, at different pH values indicated that the observed inhibition can be attributed in large part to the charged forms of the amphiphiles. Direct association of methochlorpromazine with egg phosphatidylcholine bilayers was demonstrated by molecular sieve chromatography; no such association of the amphiphile with phosphatidylinositol transfer protein was apparent. Anionic agents, such as indomethacin, phenylbutazone, and tolmetin, were without significant effect on protein-catalyzed phospholipid transfers. Electrostatic interaction between the cationic amines and anionic or zwitterionic phospholipids, forming ion pairs in the lipid bilayers, is suggested as a possible molecular mechanism for the observed inhibition.     

Partition of chlorpromazine into lipid bilayer membranes: the effect of membrane structure and composition

Partition coefficients, kp, of chlorpromazine between the aqueous phase and lipid bilayer vesicles were determined as function of drug concentration, lipid chain length, cholesterol content and temperature encompassing the range of the lipid phase transition. Radioactivity and absorption measurements were performed to determine the kp values. Up to a concentration of 3 . 10(-5) M, the partition coefficient is independent of chlorpromazine concentration, whereas it decreases drastically at higher chlorpromazine concentrations, at which membrane lysis is observed. Membrane structure is not disturbed at less than 3 . 10(-5) M chlorpromazine, as was concluded from electron paramagnetic resonance studies measuring TEMPO partitioning and order degree. However, the lipid phase-transition temperature decreases and is broadened at higher chlorpromazine concentrations. From fluorescence measurements, we conclude the formation of chlorpromazine micelles at concentrations higher than 5 . 10(-5) M in chlorpromazine in the absence of lipids and the formation of mixed micelles in the presence of lipids. The effect of lipid chain length on kp values was investigated. The partition coefficient decreases from 8100 in dilauroyl- to 3400 in dipalmitoylphosphatidylcholine vesicles, both at 50 degrees C, that is, above their corresponding phase-transition temperature tt. At t less than tt the kp values are strongly reduced, by at least a factor of 10, depending on lipid chain length and membrane composition. It is possible to establish a lipid phase-transition curve from the temperature-dependent measurements of the kp values. Cholesterol within the lipid membrane strongly decreases kp. At 20 mol% cholesterol in dipalmitoylphosphatidylcholine membranes, the partition coefficient is reduced from 3400 to 2300. This value is well comparable to the kp value obtained in erythrocyte ghosts. In contradiction to earlier experiments by Conrad and Singer (Biochemistry 20 (1981) 808-818), this value in a biological membrane could be obtained by the hygroscopic desorption as well as the centrifugation method. From our experiments we are justified in further considering artificial bilayer membranes as models for biological membranes.     

Oxygen diffusion in biological and artificial membranes determined by the fluorochrome pyrene

Quenching of pyrene fluorescence by oxygen was used to determine oxygen diffusion coefficients in phospholipid dispersions and erythrocyte plasma membranes. The fluorescence intensity and lifetime of pyrene in both artificial and natural membranes decreases about 80% in the presence of 1 atm O2, while the fluorescence excitation and emission spectra and the absorption spectrum are unaltered. Assuming the oxygen partition coefficient between membrane and aqueous phase to be 4.4, the diffusion coefficients for oxygen at 37 degrees C are 1.51 X 10(-5) cm2/s in dimyristoyl lecithin vesicles, 9.32 X 10(-6) cm2/s in dipalmitoyl lecithin vesicles, and 7.27 X 10(-6) cm2/s in erythrocyte plasma membranes. The heats of activation for oxygen diffusion are low (less than 3 kcal/degree-mol). A dramatic increase in the diffusion constant occurs at the phase transition of dimyristoyl and dipalmitoyl lecithin, which may result from an increase in either the oxygen diffusion coefficient, partition coefficient, or both. The significance of the change in oxygen diffusion below and above the phase transition for biological membranes is discussed.     

Erythrocyte ghost--buffer partition coefficients of phenobarbital, pentobarbital, and thiopental support the pH-partition hypothesis.

The partition coefficient (lambda) between red cell ghosts and buffer has been determined for three barbiturates over a range of pH. Experimental partition coefficients were linearly proportional to the calculated degree of association of the barbiturates. Lambda was 9.5 +/- 0.52 for phenobarbital, 12.7 +/- 0.91 for pentobarbital, and 27 +/- 4.9 for thiopental in their acid forms. Lambda for all three barbiturates in their anionic forms was zero. Our data support the assumption of the pH-partition hypothesis that the dependence of lambda on pH in biological membranes behaves essentially like that in organic solvents. However, the relative magnitudes of the erythrocyte partition coefficients correlate much more closely with the physiological permeability constants than do those of organic solvents, which tend to overestimate the differences between these compounds.     

Phospholipid asymmetry in human erythrocyte ghosts

Using phospholipase digestion and the fluorescent probe merocyanine 540 the maintenance of phospholipid asymmetry in the plasma membrane of human erythrocyte ghosts was investigated. Digestion with phospholipase A2 indicated that ghosts prepared in the presence of Mg++ as the only divalent cation retained the normal phospholipid asymmetry characteristic of intact erythrocytes. These ghosts, like normal erythrocytes, also failed to stain with merocyanine 540. However, the presence of as little as 5-10 microM Ca++ during ghost preparation resulted in ghosts in which lipid asymmetry had been abolished, as indicated by phospholipase digestion. Moreover, these ghosts stained with merocyanine 540. In contrast to ghosts, intact erythrocytes treated with ionophore required millimolar levels of Ca++ ions to disrupt membrane lipid asymmetry. To discover the reason for this difference in behavior between ghosts and intact cells, ghosts were prepared from preswollen cells using only small volumes of buffer for lysis. These experiments demonstrated that as the cellular contents of erythrocytes are diluted, the asymmetric arrangement of phospholipids becomes more sensitive to disruption by Ca++.     

Effect of hemoglobin A and S on human erythrocyte ghosts

The interaction of erythrocyte ghosts and vesicles with chromatographed hemoglobin (Hb) A and Hb S was studied under various conditions. Although no binding of either Hb A or Hb S to inside-out vesicles was detected, under conditions of physiological ionic strength and pH, several properties of white membrane ghosts were effected by the presence of Hb. Addition of Hb A and Hb S (2 g/dl) to membrane ghosts in 6 mM MgATP, 150 mM NaCl, 10 mM Tris-HCl buffer, pH 7.4, was found to effect the echinocyte-discocyte transition, the extent of endocytosis, the volume, and the sealing of ghosts. Our observations suggest that the structure of membrane ghosts is influenced by cytosol proteins and that the environment of the red cell membrane plays an important role in the definition and the control of the membrane structure and function.     

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.     

Preparation of inside-out vesicles from erythrocyte membranes inactivates the pathway for oleic acid incorporation into phospholipid

The pathway for membrane phospholipid fatty acid turnover in situ may be important in the regulation of the composition and turnover of the lipid microenvironment of membrane proteins. This pathway has been characterized further by studying the activation and incorporation of [9,10(n)-3H]oleic acid and transesterification of [1-14C]oleoyl-CoA into membrane phospholipids by isolated erythrocyte membrane ghosts and inside-out vesicles derived from these ghosts. Erythrocyte ghosts and sealed vesicles of defined orientation prepared from them have been widely employed in studies of the function of membrane proteins, particularly those which mediate the transport of ions and sugars. Preparation of inside-out vesicles from ghosts by exposure to alkaline hypotonic conditions results in elution of some membrane proteins but no loss of membrane phospholipid. Compared to ghosts, the ability of inside-out vesicles to activate and incorporate [9,10(n)-3H]oleic acid into phospholipid is diminished by over 90% and the ability of inside-out vesicles to transesterify [1-14C]oleoyl-CoA to phospholipid is diminished by over 50%. These findings indicate that exposure of erythrocyte membranes to the alkaline hypotonic conditions required for inside-out vesicle preparation results in loss or inactivation of both acyl-CoA ligase and acyl-CoA-lysophospholipid acyltransferase activities. This lability of the enzymes for in situ phospholipid fatty acid turnover should be considered in the design and interpretation of studies concerned with elucidation of the relationship between phospholipid fatty acid turnover and the regulation of membrane protein function in this membrane preparation.     

pKa Values and Partition Coefficients of Nitroxide Spin Probes for Membrane Bioenergetics Measurements

Knowledge of pK_a's is necessary to calculate intracellular/intravesicular pH values from nitroxide accumulation in cells or vesicles as detected with electron spin resonance (ESR) spectroscopy. pK_a values were confirmed in lipid vesicles of known internal pH. To help select probes that do not accumulate in lipid membranes, octanol/buffer partition coefficients of uncharged nitroxides were determined. As an application of selected probes, pH gradients and internal aqueous volumes were analyzed in mitochondria (one internal compartment) and in the cyanobacterium Synechococcus 6311 (two internal compartments). The combination of 3-carboxy-, 3-amino- and 3-aminocarbonyl-2,2,5,5-tetramethylpyrrolidin-1-yloxyl was found to be most satisfactory for determinations of internal pH and volumes.     

ESR spectral changes induced by chlorpromazine in spin-labeled erythrocyte ghost membranes

chlorpromazine interacted preferentially with membrane proteins rather than membrane lipids in the initial incorporation into human erythrocyte ghosts, as demonstrated by means of the fluorescence quenching and a maleimide spin label. In this state the membrane fluidity increased. At higher concentrations of chlorpromazine, the membrane fluidity decreased and a motionally restricted signal from fatty acid spin labels appeared predominantly. However, no such signal appeared in protein-free vesicles. The temperature and pH dependences of the outer hyperfine splitting of this restricted signal were very similar to those of bovine serum albumin. On the basis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of chlorpromazine-treated and -untreated ghosts, it was found that there was no significant difference in membrane proteins between both samples except for the changes of a few bands which were not directly concerned with the occurrence of this restricted signal. These results suggest that the fatty acid spin labels bind preferably to membrane proteins as the lipid domain becomes packed with chlorpromazine.     

Partition of piperidinoethylesters of 2-alkyloxyphenylcarbamic acid in unilamellar phosphatidylcholine liposomes.

Molar partition coefficients for amphiphilic N-[2-(2-alkyl-oxyphenyl-carbamoyloxy)-ethyl]-piperidinium chlorides (PAA) between small unilamellar egg yolk phosphatidyl choline liposomes and saline, as determined by ultraviolet difference spectroscopy at 22 degrees C, pH 5-6, v = 34640 cm-1, and at 100 mumol/l PAA concentration, were 149, 1990, and 7474 for PAA with 5, 7, and 9 carbon atoms in the alkyloxy substituent, respectively. At the PAA concentration used, the cut-off in biological activities of PAAs with long alkyloxy substituents could not be caused by the self-association of PAA molecules in the aqueous phase.     

Kinetics and thermodynamics of association of a phospholipid derivative with lipid bilayers in liquid-disordered and liquid-ordered phases

We have measured the rates of insertion into, desorption from, and spontaneous interlayer translocation (flip-flop) in liquid-disordered and liquid-ordered phase lipid bilayer membranes, of the fluorescent phospholipid derivative NBD-dimyristoylphosphatidyl ethanolamine. This study made use of a recently described method that exploits a detailed knowledge of the binding kinetics of an amphiphile to bovine serum albumin, to recover the insertion and desorption rate constants when the albumin-bound amphiphile is transferred through the aqueous phase to the membrane and vice versa. The lipid bilayers, studied as large unilamellar vesicles, were prepared from pure 1-palmitoyl-2-oleoylphosphatidylcholine in the liquid-disordered phase; and from two cholesterol-containing binary lipid mixtures, 1-palmitoyl-2-oleoylphosphatidylcholine and cholesterol (molar ratio of 1:1), and egg sphingomyelin and cholesterol (molar ratio of 6:4), both in the liquid-ordered phase. Insertion, desorption, and translocation rate constants and equilibrium constants for association of the amphiphile monomer with the lipid bilayers were directly measured between 15 degrees and 35 degrees C, and the standard free energies, enthalpies, and entropies, as well as the activation energies for these processes, were derived from this data. The equilibrium partition coefficients for partitioning of the amphiphile between the aqueous phase and the different membrane phases were also derived, and permitted the estimation of hypothetical partition coefficients and the respective energetic parameters for partitioning between the different lipid phases if these were to coexist in the same membrane.     

Dependency of delta pH-relaxation across vesicular membranes on the buffering power of bulk solutions and lipids.

The dependency of delta pH-relaxation kinetics across the membrane of sonicated small phospholipid vesicles on the concentration of internally entrapped buffer has been investigated by means of the pH-indicator dye pyranine. A very high contribution of lipid headgroups to the internal buffering power of the liposomes is observed, amounting to an equivalent phosphate buffer concentration of 110 mM. This localized two-dimensional proton/hydroxide ion reservoir must be considered in any determination of the H+/OH- permeability coefficient. Furthermore, it could have significance for energy-transduction across biological membranes. From the established linear relation between delta pH-relaxation rates and buffering power, net H+/OH- permeabilities of 3 X 10(-3) cm/s for soybean phospholipid (SBPL) and 1 X 10(-4) cm/s for diphytanoyl phosphatidylcholine (diphytanoyl PC) vesicles at pH 7.2 as well as buffering powers per lipid molecule of 6 X 10(-2) (pH-unit)-1 (SBPL) and 4 X 10(-2) (pH-unit)-1 (diphytanoyl PC) are calculated. In the case of diphytanoyl PC vesicles, delta pH-decay is accelerated by the presence of chloride ions.     

On the water and proton permeabilities across membranes from erythrocyte ghosts

The diffusional permeability of water across membranes from bovine and human erythrocyte ghosts was measured by a recently developed method which is based on the different indices of refraction of H2O and 2H2O. Resealed erythrocyte ghosts were prepared by a gel-filtration technique. Pd (2H2O/H2O) values of 1.2 X 10(-3) cm/s (human) and 1.7 X 10(-3) cm/s (bovine) were calculated at 20 degrees C. The activation energies of the water exchange were 23.5 kJ/mol (human) and 25.4 kJ/mol (bovine). Treatment of the ghosts with p-chloromercuribenzenesulfonic acid (PCMBS) led to a 60-70% inhibition of the diffusional water exchange. The pH equilibration across membranes of erythrocyte ghosts was measured by intracellular carboxyfluorescein. The rates of proton flux after pH-jumps (pH 7.3 to pH 6.1) were about 100-fold lower than those of the water exchange and dependent on the kind of anions present (Cl-, NO-3, SO2-4). The activation energies of proton flux were 60-70 kJ/mol. 4,4'-Diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) inhibited the exchange by 97-98% and lowered the activation energy. The inhibitor of water exchange, PCMBS, increased the proton-permeation rate by a factor of 4-5. It is assumed that the rate-limiting step for the proton permeation is determined by the anion exchange. Under this condition our results are not in accord with one channel as a common pathway for both the passive water and anion transport.     

Phospholipid lateral phase separation and the partition of cis-parinaric acid and trans-parinaric acid among aqueous, solid lipid, and fluid lipid phases.

The partition of cis-parinaric acid (9,11,13,15-cis, trans, trans,cis-octadecatetraenoic acid, cis-PnA) and trans-parinaric acid (9,11,13,15-all-trans-octadecatetraenoic acid, trans-PnA) among aqueous, solid lipid, and fluid lipid phases has been measured by three spectroscopic parameters: absorption spectral shifts, fluorescence quantum yield, and fluorescence polarization. The solid lipid was dipalmitoylphosphatidylcholine (DPPC); the fluid lipid was palmitoyldocosahexaenoylphosphatidylcholine (PDPC). Mole fraction partition coefficients between lipid and water were determined by absorption spectroscopy to be for ci--PnA, 5.3 X 10(5) with a solid lipid and 9 X 10(5) with fluid lipid and, for trans-PnA, 5 X 10(6) with solid lipid and 1.7 X 10(6) with fluid lipid. Ratios of the solid to the fluid partition coefficients (Kps/f) are 0.6 +/- 0.2 for cis-PnA and 3 +/- 1 for trans-PnA. A phase diagram for codispersions of DPPC and PDPC has been constructed from the measurements of the temperature dependence of the fluorescence quantum yield and polarization of cis-PnA and trans-PnA and their methyl ester derivatives. A simple analysis based on the phase diagram and fluorescence data allows additional calculations of Kps/f's which are determined to be 0.7 +/- 0.2 for the cis probes and 4 +/- 1 for the trans probes. The relative preference of trans-PnA for solid phase lipids and its enhanced quantum yield in solid phase lipids make it sensitive to a few percent solid. The trans probes provide evidence that structural order may persist in dispersions of these phospholipids 10 degrees C or more above their transition temperature. It is concluded that measurements of PnA fluorescence polarization vs. temperature are better suited than measurements of quantum yield vs. temperature for determining phospholipid phase separation.     

ATP-induced endocytosis in human erythrocyte ghosts. Characterization of the process and isolation of the endocytosed vesicles.

ATP-induced endocytosis in human erythrocyte ghosts has been studied, and a procedure for the isolation of the endocytotic vesicles is described. Under isotonic conditions and 37 degrees C, optimal endocytosis occurs with concentrations of 4 to 10 mM MgATP. Within 30 min, up to 45% of the membrane is removed from the surface and converted into sealed inside-out vesicles. Local anesthetics, such as chlorpromazine, potentiate ATP-induced endocytosis in ghosts. Forcing cells containing endocytotic vesicles through a hypodermic needle leads to the exclusive fragmentation of the outermost plasma membrane. The endocytosed vesicles can then be separated from these fragments by centrifugation on a gradient of dextran T70. Biochemical analyses indicate that endocytotic vesicles contain full complements of the major membrane proteins (i.e. also spectrin and actin), common phospholipids, fatty acids, and cholesterol. Furthermore, they exhibit a fully intact spectrin component 2 phosphorylation machinery. In contrast, MgATPase activity is largely excluded from these vesicles. The novel inside-out vesicles described have properties different from those of previously analyzed fragments of the erythrocyte membrane. They will permit a detailed study of a native spectrin-actin network now exposed to the outside.     

The displacement of phenothiazines from phospholipid binding sites by cholesterol

The interaction of the phenothiazine type drug, methochlorpromazine, with phosphatidylcholine membranes has been investigated by using this tranquilizer in a deuterium labeled form. Two distinct binding sites were found, with exchange between them being fast on the 2H NMR time scale. Cholesterol preferentially displaces the chlorpromazine from the more hydrophobic of these sites, making possible an explanation of the modulation of the effects of amphipathic agents by cholesterol. In addition, the phenomenon of displacement of membrane active agents by sterols may explain discrepancies between membrane/water partition coefficients as measured by centrifugation and hygroscopic desorption.     

ESR spectral changes induced by chlorpromazine in spin-labeled erythrocyte ghost membranes

Chlorpromazine interacted preferentially with membrane proteins rather than membrane lipids in the initial incorporation into human erythrocyte ghosts, as demonstrated by means of the fluorescence quenching and a maleimide spin label. In this state the membrane fluidity increased. At higher concentrations of chlorpromazine, the membrane fluidity decreased and a motionally restricted signal from fatty acid spin labels appeared predominantly. However, no such signal appeared in protein-free vesicles. The temperature and pH dependences of the outer hyperfine splitting of this restricted signal were very similar to those of bovine serum albumin. On the basis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of chlorpromazine-treated and -untreated ghosts, it was found that there was no significant difference in membrane proteins between both samples except for the changes of a few bands which were not directly concerned with the occurrence of this restricted signal. These results suggest that the fatty acid spin labels bind preferably to membrane proteins as the lipid domain becomes packed with chlorpromazine.     

Binding of Cerebratulus cytolysin A-III to human erythrocyte membranes

Binding of Cerebratulus lacteus cytolysin A-III to intact human erythrocytes and erythrocyte membranes has been investigated. Binding to ghosts is essentially complete within 2.5 min of mixing which is slightly faster than the rate of hemolysis measured with intact cells. Approximately 4 X 10(4) binding sites per cell, exhibiting a K 0.5 of 0.7 microM exist; this compares with 50% hematocrit of about 0.3 microM for A-III. Binding is absent in ghosts extracted with Nonidet P-40, but is unaffected by pretreatment of ghosts with either trypsin or elastase.