An ultrarapid filtration method adapted to the measurements of water and solute permeability of synthetic and biological vesicles.

An ultrarapid filtration method was adapted to the determination of water and solute permeability of membrane vesicles. This method consisted of measuring substance washout from vesicles first loaded with 3H2O or labeled solutes, placed on filters, and rinsed at high rates for short periods. The retention of the vesicles on the filters was analyzed and was found to be a function of the nature and porosity of the filters as well as of the vesicle origin. Washing buffer flow rate and washing duration did not affect vesicle retention. The diffusional water permeability of cholesterol-free liposomes was determined at 16 degrees C. Its value was reduced by a factor of 2.5 when the liposomes were prepared with 20% cholesterol and a threefold increase was noted when the liposomes were preincubated with gramicidin (6 mg/g lipid). Water permeability of liposomes was strongly temperature-dependent: Ea = 15.3 kcal/mol. Diffusional water permeability of pink ghosts was also measured: a value of (4.4 +/- 0.2) X 10(-3) cm/s (n = 3) was obtained at 13 degrees C. This permeability was reduced by 45.2% with 0.4 mM HgCl2. The urea permeability of intestinal and renal brush-border membrane vesicles was (1.15 +/- 0.18) X 10(-6) cm/s (n = 7) and (1.67 +/- 0.08) X 10(-6) cm/s (n = 9), respectively. The renal value was reduced by a factor of 4.4 by 100 mM thiourea. This ultrarapid filtration technique provides an accurate method of transport measurement in sealed membranes such as liposomes and plasma membrane vesicles.     

Transmembrane calcium movements mediated by ionomycin and phosphatidate in liposomes with Fura 2 entrapped

A novel liposomal method permits studies of Ca movements across the bilayers of multilamellar vesicles (MLV) which had entrapped the Ca-dependent, fluorescent indicator dye Fura 2. Ionomycin-mediated Ca translocation across MLV of phosphatidylcholine (PC)/dicetyl phosphate (DCP), 9:1, obeyed simple first-order kinetics since log-log plots of initial rates versus ionomycin or Ca concentration yielded slopes of approximately 1. Since Ca is translocated in a Ca-dependent fashion in the course of stimulus-response coupling of cells which form diacylglycerol (DAG) and phosphatidate (PA) from polyphosphoinositides, we compared effects of PA with those of DAG. PA and DAG were preincorporated in PC/DCP vesicles, in which trace amounts of ionomycin provided transmembrane potential (due to Ca2+/H+ exchange). Significant increases in Ca movements were observed in the presence of egg lecithin PA, dioleoyl-PA, and dipalmitoyl-PA when compared with DCP- or DAG-containing MLV. DAGs such as 1-oleoyl-2-acetoylglycerol or 1,2-dioleoylglycerol in liposomes decreased rates of Ca translocation. Ca influx into PA-containing MLV was dependent on the mole percent of the PA in bilayers; the complex kinetics of Ca influx were compatible with the formation of nonbilayer states. Incorporation of cholesterol into the liposomes inhibited initial rates of Ca uptake by MLV presumably by condensing the bilayers. Ca influx increased with increasing pH of the external medium from 6.9 to 7.9 in liposomes with an internal pH of 7.4.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ion selectivity of temperature-induced and electric field induced pores in dipalmitoylphosphatidylcholine vesicles

Temperature and electric field are known to alter the permeability of the bilayer membrane in phospholipid vesicles. A study of cation selectivity of these membrane pores is reported for multilamellar liposomes (MLV) and unilamellar large vesicles (ULV, 95 +/- 5 nm diameter) of dipalmitoylphosphatidylcholine (DPPC). The permeability of ULV to Rb+ was 1.0 X 10(-6) micrograms/s at 22 degrees C and increased to 1.1 X 10(-5) micrograms/s at the gel to liquid-crystalline transition temperature (Tm) of the bilayer, at 42 degrees C. The permeability of ULV to Rb+ continued to increase beyond the Tm and reached 1.0 X 10(-4) micrograms/s at 56 degrees C, a 100-fold increase over the permeability at 22 degrees C. In contrast, the permeability of ULV to Na+ showed a local maximum of 6.0 X 10(-6) micrograms/s at 42 degrees C and decreased at temperatures higher or lower than the Tm. For MLV, the permeability to both Rb+ and Na+ peaked dramatically at the phase transition temperature, 42 degrees C, and subsided at lower and higher temperatures. When ULV were exposed to an electric field, the permeability to Rb+, Na+, and sucrose surged at a field strength of 30 kV/cm; 30 kV/cm can induce a transmembrane potential of 210 mV. In ULV, the electrically perforated lipid bilayer exhibited selectivity for Rb+ over Na+ only at a narrow electric field range, between 31 and 33 kV/cm. For MLV, no well-defined breakdown voltage was recorded.(ABSTRACT TRUNCATED AT 250 WORDS)     

Preparation and characterization of reactive and stable glucose oxidase-containing liposomes modulated with detergent

Glucose oxidase-containing liposomes (GOL) as well as detergent-modulated glucose oxidase-containing liposomes were prepared and characterized, focusing not only on the reactivity of the liposomes upon external addition of glucose but also on the leakage of the entrapped glucose oxidase (GO) from the liposomes with the aim of developing a reactive and stable liposomal GO system. The membranes of the GOL prepared were composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and modulated with either Triton X-100 or cholate. In the absence of added detergent, no GO leakage from the GOL was observed while its enzymatic activity was very low (low glucose permeability). As detergent-modulated liposomes, mixed POPC/Triton X-100 and mixed POPC/cholate liposomes (abbreviated as TL and CL, respectively) were prepared at different effective detergent/POPC molar ratios (R(e)) ranging from R(e) = 0 to R(e) = R(e) (sat) (R(e) (sat) is the critical value of R(e) at which the liposome membrane is saturated with detergent). The reactivity of GO-loaded TL (abbreviated as GOTL) or GO-loaded CL (GOCL) increased drastically with increase in the respective detergent content in the liposomes. In the case of GOTL, at R(e) (sat) = 0.40, a high reactivity was measured with a simultaneous high extent of GO leakage, suggesting that the observed enzymatic reaction was catalyzed mainly by leaked GO, caused by the interaction of Triton X-100 with the POPC membrane. On the other hand, GOCL prepared at R(e) (sat) = 0.43 showed relatively high reactivity with only a small extent of GO leakage, suggesting that most of the enzyme reaction was limited by the glucose permeation across the bilayers of GOCL. The GO leakage from GOCL was found to occur mostly during the rearrangement of the liposomal membrane during the preparation of the GOCL (mixing the GOL and cholate). Fluorescence polarization measurements of membrane-associated DPH (1,6-diphenyl-1,3,5-hexatriene) indicated that CL prepared by modifying POPC with cholate did not lead to a drastic change in membrane fluidity, indicating that the interacting cholate molecules did not penetrate deeply into the POPC bilayers. In summary, it was clearly shown that the membrane permeability of GOL can be quite simply modulated by mixing it with a certain amount of cholate to form highly reactive and stable GOCL with minimal enzyme leakage.     

Membrane-mediated assembly of the prothrombinase complex

Prothrombinase assembly was studied on macroscopic planar bilayers consisting of 20% dioleoyl-phosphatidylserine (DOPS) and 80% dioleoyl-phosphatidylcholine (DOPC). The dissociation constant for the binding of factor Xa to the bilayer, measured by ellipsometry, was Kd = 47 +/- 8 nM (mean +/- S.D.) and this value was lowered to Kd = 2.2 +/- 0.3 pM by preadsorption of factor Va. This latter value was determined from direct measurement of steady-state thrombin production. A comparable value of Kd = 1.0 +/- 0.1 pM was found by repeating these experiments in suspensions of phospholipid vesicles, and it was verified that prothrombinase assembly was not influenced by the addition of prothrombin. Using a minute amount (0.094 fmol cm-2) of preadsorbed factor Va, it was found that the rate of prothrombinase assembly exceeds the rate of collisions between Xa molecules from the buffer and the sparse Va molecules on the bilayer. Apparently, factor Xa adsorbs first to the membrane and then associates rapidly with factor Va by lateral diffusion. The data indicate almost instantaneous equilibrium of this complex formation on the surface with a lower limit for the bimolecular rate constant of kon = 2.8 x 10(13) (mol/cm2)-1 s-1. In suspensions of small phospholipid vesicles, prothrombinase assembly is collisionally limited and the value of kon should be proportional to vesicle diameter. This was verified with a method for estimation of kon values from thrombin generation curves. Values of 0.36 x 10(9) and 1.6 x 10(9) M-1 s-1 were found for vesicles of 20-30- and 60-80-nm diameter, respectively.     

Electric field induced transient pores in phospholipid bilayer vesicles

A study of the voltage induction of transient pores in phospholipid bilayer vesicles is reported. Unilamellar vesicles (dipalmitoylphosphatidylcholine), with a size distribution of 100 +/- 30 nm, were prepared by the method of Enoch & Strittmatter [Enoch, H., & Strittmatter, P. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 145]. The vesicles loaded with [14C]sucrose and suspended in a mixture of 150 mM NaCl and 272 mM sucrose (both are the isotonic solvent for erythrocytes) were exposed to an intense electric field in the range of 20--40 kV/cm, with a field decay time of 5--15 micro second. A transient leakage of sucrose label was detected when the field strength exceeded 30 kV/cm. After the field was removed, no slow leakage of the tracer molecules occurred during a 65-h incubation period at the room temperature (23 +/- 2 degrees C). The leakage is attributed to the field-induced transmembrane potential, but not other effects such as the Joule heating or the shock wave associated with the voltage discharge. When the potential exceeded a threshold value of 200 mV, corresponding to an applied field strength of 30 kV/cm, there was a dielectric breakdown of the bilayer structure. Pores which allowed passage of sucrose were formed, transiently. Experiments show that these pores were fully reversible, and no global and permanent damages to the vesicle bilayer were detected. The implication of this membrane potential triggered conducting state of lipid bilayers to biological functions of cells is discussed.     

Response of isolated sperm plasma membranes from sea urchin to egg jelly

The acrosome reaction in sea urchin sperm is induced by a glycoprotein jelly surrounding the egg and is accompanied by changes in ion permeability of sperm plasma membrane. In an attempt to learn what membrane components are involved in the response to jelly, we have begun to reassemble sperm membrane components into artificial membranes and assay for permeability changes mimicking those that occur in sperm. Jelly in sea water at concentrations that induce the acrosome reaction did not significantly change 45Ca2+ uptake of sonicated unilamellar vesicles made with soybean lipid only (ratio jelly:control uptake = 1.08 +/- 0.36 SD, n = 21). Experiments with pure lipid planar bilayers made with soybean lipid or a lipid extract from sperm and held at various voltages, also did not reveal substantial permeability changes at comparable jelly concentrations. Thus, jelly by itself does not change the conductance of a pure lipid bilayer. In contrast, significant (P----0.0005, t test for two sample means) 45Ca2+ uptake was observed with vesicles made by cosonicating soybean phospholipids and Strongylocentrotus purpuratus sperm membranes isolated by the method of Cross, N. L. [1983, J. Cell Sci. 59, 13-25] (ratio jelly: control uptake = 1.51 +/- 0.75, n = 20, 16 positive out of 20 experiments). The calcium uptake response of the mixed vesicles was also species-specific: it did not occur with jelly from Arbacia punctulata (ratio Arbacia jelly: control = 1.18 +/- 0.51; ratio Strongylocentrotus jelly: control = 1.71 +/- 0.97, n = 10; P----0.025, paired t statistic). Vesicles made with soybean lipid and an octyl glucoside extract of sperm membranes also responded to jelly with increased 45Ca2+ uptake. Our results indicate that we have the starting conditions to isolate and characterize the sperm membrane components that participate in the egg jelly induced permeability changes.     

Effects of cholesterol and temperature on the permeability of dimyristoylphosphatidylcholine bilayers near the chain melting phase transition

The passive leakage of glucose across bilayers of dimyristoylphosphatidylcholine (DMPC), cholesterol (variable), and dicetyl phosphate (constant 5.9 mol%) has been measured as efflux over 30 min from multilamellar vesicles. Bilayer cholesterol was varied from 20 mol% to 40 mol%. Glucose permeation rates were measured from 10 degrees C to 36 degrees C, and showed a maximum in permeability at 24 degrees C, the DMPC phase transition temperature. Increasing the bilayer cholesterol content above 20 mol% reduced that permeability peak. These results are quite consistent with a large number of similar bilayer permeability studies over the past 25 years. However, they are not consistent with a previous study of these same systems, which reported increased glucose permeability with temperature, without any maximum at or near the lipid chain melting temperature (K. Inoue, Biochim. Biophys. Acta 339 (1974) 390-402).     

Physicochemical characterization of large unilamellar phospholipid vesicles prepared by reverse-phase evaporation

Properties of large unilamellar vesicles (LUV), composed of phosphatidylcholine and prepared by reverse-phase evaporation and subsequent extrusion through Unipore polycarbonate membranes, have been investigated and compared with those of small unilamellar vesicles (SUV) and of multilamellar vesicles (MLV). The unilamellar nature of the LUV is shown by 1H-NMR using Pr3+ as a shift reagent. The gel to liquid-crystalline phase transition of LUV composed of dipalmitoylphosphatidylcholine (DPPC) monitored by differential scanning calorimetry, fluorescence polarization of diphenylhexatriene and 90 degrees light scattering, occurs at a slight lower temperature (40.8 degrees C) than that of MLV (42 degrees C) and is broadened by about 50%. The phase transition of SUV is shifted to considerably lower temperatures (mid-point, 38 degrees C) and extends over a wide temperature range. In LUV a well-defined pretransition is not observed. The permeability of LUV (DPPC) monitored by leakage of carboxyfluorescein, increases sharply at the phase transition temperature, and the extent of release is greater than that from MLV. Leakage from SUV occurs in a wide temperature range. Freeze-fracture electron microscopy of LUV (DPPC) reveals vesicles of 0.1-0.2 micron diameter with mostly smooth fracture faces. At temperatures below the phase transition, the larger vesicles in the population have angled faces, as do extruded MLV. A banded pattern, seen in MLV at temperatures between the pretransition and the main transition, is not observed in the smaller LUV, although the larger vesicles reveal a dimpled appearance.     

Production of thrombin by the prothrombinase complex is regulated by membrane-mediated transport of prothrombin

Production of thrombin by phospholipid-bound prothrombinase complexes has been described as being regulated by the prothrombin concentration in the buffer (free-substrate model) as well as by the concentration of prothrombin adsorbed to the phospholipid surface (bound-substrate model). We studied simultaneous adsorption and conversion of prothrombin on planar bilayers consisting of 20% dioleoylphosphatidylserine and 80% dioleoylphosphatidylcholine. A transport limitation in the conversion of prothrombin was prevented by using a very low (0.3 fmol cm-2) amount of prothrombinase on the bilayer. The Michaelis and catalytic constants thus found were Km = 5.8 +/- 0.7 nM and kcat = 33 +/- 1 s-1 (mean +/- S.D.). The apparent bimolecular rate constant Kcat/Km = 5.7 x 10(9) M-1 s-1 exceeds the theoretically maximal value for the free-substrate model. In contrast, kcat/Km is within the range expected for a diffusion-controlled bound-substrate model. A similar mechanism for prothrombin conversion in suspensions of phospholipid vesicles would imply increasing kcat/Km values for increasing vesicle diameter. This prediction was tested and a 3-fold increase in kcat/Km values was indeed found for vesicles 60-80 nm in diameter compared to vesicles of 20-30 nm diameter. It is concluded that thrombin production is dependent on protein fluxes rather than on protein concentrations.     

Vasopressin-enhanced urea transport by rat inner medullary collecting duct cells in culture

The distal inner medullary collecting duct (IMCD) is critical in the urinary concentrating process, in part because it is the site of vasopressin (AVP)-regulated permeability to urea. The purpose of these experiments was to develop a cell culture model of the IMCD on permeable structure and to characterize the responsiveness to AVP. Rat IMCD cells were grown to confluence on collagen-coated Millipore filters glued onto plastic rings. To assess the time required to achieve confluence, the transepithelial resistance was measured periodically and was found to be stable after 2 weeks, at a maximal value of 595 +/- 22 omega cm2. In separate monolayers the effect of AVP on inulin and urea permeability was determined. While inulin permeability was unchanged after AVP, urea permeability increased from 6.0 +/- 0.4 to peak values of 16.0 +/- 3.8 (10 nM), 23.1 +/- 3.9 (1 microM) and 28.1 +/- 4.9 (10 microM) x 10(-6) cm s-1 (n = 24). In 10 other monolayers, after the addition of 1 mM 8-Br-cAMP, urea permeability increased from 5.1 +/- 0.3 to 8.1 +/- 1.6 x 10(-6) cm s-1 and, after 8-Br-cAMP + 3-isobutyl-1-methylxanthine, to 12.2 +/- 0.7 x 10(-6) cm s-1. We conclude that rat IMCD cells grown in culture exhibit the characteristics of a 'tight' epithelium. Inulin and urea permeability are not different in the absence of AVP, consistent with high resistance junctional complexes. Furthermore, IMCD cells retain the capacity for AVP-regulated urea permeability, a characteristic feature of this nephron segment in vivo.     

Electrogenic H+/OH- movement across phospholipid vesicles measured by spin-labeled hydrophobic ions.

Transmembrane pH gradients created across phospholipid vesicles give rise to time-dependent potentials as determined from the EPR spectra of phosphonium ion spin labels in the system. From the time-dependent spectra, the transmembrane H+/OH- current is obtained and hence the current-voltage curve for the vesicle membrane is obtained. The current-voltage curve is linear with a membrane resistance of 3 +/- 2 X 10(9) omega cm2 corresponding to a membrane permeability of 5 +/- 2 X 10(-7) cm/s. This unusually high permeability is further increased by small amounts of lipid oxidation, CHCl3 or the general anesthetic halothane.     

Water permeability of asymmetric planar lipid bilayers: leaflets of different composition offer independent and additive resistances to permeation

To understand how plasma membranes may limit water flux, we have modeled the apical membrane of MDCK type 1 cells. Previous experiments demonstrated that liposomes designed to mimic the inner and outer leaflet of this membrane exhibited 18-fold lower water permeation for outer leaflet lipids than inner leaflet lipids (Hill, W.G., and M.L. Zeidel. 2000. J. Biol. Chem. 275:30176-30185), confirming that the outer leaflet is the primary barrier to permeation. If leaflets in a bilayer resist permeation independently, the following equation estimates single leaflet permeabilities: 1/P(AB) = 1/P(A) + 1/P(B) (Eq. l), where P(AB) is the permeability of a bilayer composed of leaflets A and B, P(A) is the permeability of leaflet A, and P(B) is the permeability of leaflet B. Using for the MDCK leaflet-specific liposomes gives an estimated value for the osmotic water permeability (P(f)) of 4.6 x 10(-4) cm/s (at 25 degrees C) that correlated well with experimentally measured values in intact cells. We have now constructed both symmetric and asymmetric planar lipid bilayers that model the MDCK apical membrane. Water permeability across these bilayers was monitored in the immediate membrane vicinity using a Na+-sensitive scanning microelectrode and an osmotic gradient induced by addition of urea. The near-membrane concentration distribution of solute was used to calculate the velocity of water flow (Pohl, P., S.M. Saparov, and Y.N. Antonenko. 1997. Biophys. J. 72:1711-1718). At 36 degrees C, P(f) was 3.44 +/- 0.35 x 10(-3) cm/s for symmetrical inner leaflet membranes and 3.40 +/- 0.34 x 10(-4) cm/s for symmetrical exofacial membranes. From, the estimated permeability of an asymmetric membrane is 6.2 x 10(-4) cm/s. Water permeability measured for the asymmetric planar bilayer was 6.7 +/- 0.7 x 10(-4) cm/s, which is within 10% of the calculated value. Direct experimental measurement of P(f) for an asymmetric planar membrane confirms that leaflets in a bilayer offer independent and additive resistances to water permeation and validates the use of.     

Lipid-polyethylene glycol interactions: II. Formation of defects in bilayers

Summary Polyethylene glycol, a known cell fusogen, is found to induce the formation of structural defects in egg phosphatidylcholine multilamellar vesicles, as shown by freeze-fracture microscopy.³¹P NMR spectra of these vesicles reveal the existence of a nonbilayer (isotropic) phase. The observed disruption in the bilayers is believed to be associated with an intermediate stage of membrane fusion.Abbreviations PEG Polyethylene glycol - IMP Intramembranous particle - PC Phosphatidylcholine - PS Phosphatidylserine - SUV Small unilamellar vesicles - MLV Multilamellar vesicles - DPPC Dipalmitoyl phosphatidylcholine - DSC Differential scanning calorimetry - DMPC Dimyristoylphosphatidylcholine - T _c Phase transition temperature     

Role of glucose carrier in human erythrocyte water permeability.

Although the transport properties of human erythrocyte water channels have been well characterized, the identity of the protein(s) mediating water flow remains unclear. Recent evidence that glucose carriers can conduct water raised the possibility that the glucose carrier, which is abundant in human erythrocytes, is the water channel. To test this possibility, water permeabilities and glucose fluxes were measured in large unilamellar vesicles (LUV) containing human erythrocyte lipid alone (lipid LUV), reconstituted purified human erythrocyte glucose carrier (Glut1 LUV), or reconstituted glucose carrier in the presence of other human erythrocyte ghost proteins (ghost LUV). In glucose and ghost LUV, glucose carriers were present at 25% of the density of native erythrocytes, were oriented randomly in the bilayer, and exhibited characteristic inhibition of glucose flux when exposed to cytochalasin B. Osmotic water permeability (Pf, in centimeters per second; n = 4) averaged 0.0012 +/- 0.00033 in lipid LUV, 0.0032 +/- 0.0015 in Glut1 LUV, and 0.006 +/- 0.0014 in ghost LUV. Activation energies of water flow for the three preparations ranged between 10 and 13 kcal/mol; p-(chloromercuri)benzenesulfonate (pCMBS), an organic mercurial inhibitor of erythrocyte water channels, and cytochalasin B did not alter Pf. These results indicate that reconstitution of glucose carriers at high density increases water permeability but does not result in water channel activity. However, because the turnover number of reconstituted carriers is reduced from that of native carriers, experiments were also performed on erythrocyte ghosts with intact water channel function. In ghosts, Pf averaged 0.038 +/- 0.013 (n = 9), while the activation energy for water flow averaged 3.0 +/- 0.3 kcal/mol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enzymatic reaction in a vesicular microreactor: peptaibol-facilitated substrate transport

Catalytic reactions performed with enzymes localized in lipid vesicles or in whole cells represent a new, promising approach in biocatalysis. The delivery of different substrates into these micro- or nano-'reactors' requires a sufficient permeability of lipid membranes. To increase the permeability of lipid bilayers, one may use different membrane-active peptides, including peptaibols. In the present study, the trypsin-catalyzed hydrolysis of N(alpha)-benzoyl-L-arginine-para-nitroanilide (BAPA; 1) was studied in a phospholipid vesicular system made of phosphatidylcholine (POC), in the presence of the peptaibols alamethicin (ALM) or zervamicin IIB (ZER). Two different manners of compartmentalization of substrate and enzyme (enzyme- vs. substrate-containing vesicles) were used. The kinetics parameters of the reaction in homogeneous solution and in the vesicular systems were determined. The rate of the extra- or intravesicular enzymatic reaction was found to be controlled by substrate diffusion through the lipid bilayer. In comparison with untreated vesicular systems, an up to seven-fold increase in reaction rate was observed in the presence of either ALM or ZER.     

Transport methods for probing the barrier domain of lipid bilayer membranes.

Two experimental techniques have been utilized to explore the barrier properties of lecithin/decane bilayer membranes with the aim of determining the contributions of various domains within the bilayer to the overall barrier. The thickness of lecithin/decane bilayers was systematically varied by modulating the chemical potential of decane in the annulus surrounding the bilayer using different mole fractions of squalene in decane. The dependence of permeability of a model permeant (acetamide) on the thickness of the solvent-filled region of the bilayer was assessed in these bilayers to determine the contribution of this region to the overall barrier. The flux of acetamide was found to vary linearly with bilayer area with Pm = (2.9 +/- 0.3) x 10(-4) cm s-1, after correcting for diffusion through unstirred water layers. The ratio between the overall membrane permeability coefficient and that calculated for diffusion through the hydrocarbon core in membranes having maximum thickness was 0.24, suggesting that the solvent domain contributes only slightly to the overall barrier properties. Consistent with these results, the permeability of acetamide was found to be independent of bilayer thickness. The relative contributions of the bilayer interface and ordered hydrocarbon regions to the transport barrier may be evaluated qualitatively by exploring the effective chemical nature of the barrier microenvironment. This may be probed by comparing functional group contributions to transport with those obtained for partitioning between water and various model bulk solvents ranging in polarity or hydrogen-bonding potential. A novel approach is described for obtaining group contributions to transport using ionizable permeants and pH adjustment. Using this approach, bilayer permeability coefficients of p-toluic acid and p-hydroxymethyl benzoic acid were determined to be 1.1 +/- 0.2 cm s-1 and (1.6 +/- 0.4) x 10(-3) cm s-1, respectively. From these values, the -OH group contribution to bilayer transport [delta(delta G0-OH)] was found to be 3.9 kcal/mol. This result suggests that the barrier region of the bilayer does not resemble the hydrogen-bonding environment found in octanol, but is somewhat less selective (more polar) than a hydrocarbon solvent.     

Ca2+-induced phosphatidylcholine vesicle aggregation in the presence of ferricyanide

The titration of sonicated vesicles of egg phosphatidylcholine with ferricyanide in the presence of Ca2+ results in the formation of aggregates. The turbidity increase caused by these aggregates cannot be reversed by EDTA treatment. In addition, no rearrangement of the bilayer structure has been found in this process, either measuring leakage of vesicle content or exchange of lipids among the bilayers themselves. The aggregation is dependent on the Ca2+ content of the vesicles, the outer Ca2+ and Fe(CN)3-(6) concentration and the order of addition of Ca2+ and ferricyanide. The results can be explained by a specific adsorption of Fe(CN)3-(6) to bilayers of sonicated vesicles, in contrast to other multivalent anions. In contrast to the stability found with sonicated vesicles, the aggregation causes a leakage of the internal solution when multilamellar liposomes are titrated with Fe(CN)3-(6).     

Mechanism of proton permeation through chloroplast lipid membranes.

Electrical measurements were carried out to investigate the contribution of chloroplast lipids to the passive proton permeability of both the thylakoid and inner-envelope membranes. Permeability coefficient and conductance to protons were measured for solvent-free bilayers made from monogalactosyldiglyceride:digalactosyldiglycerid: sulfoquinovosyldiglyceride:phosphatidylglycerol (2:1:0.5:0.5, w/w) in the presence of a pH gradient of 7.4/8.1. The permeability coefficient for protons in glycolipids was 5.5 +/- 1.1 x 10(-4) cm s-1 (n = 14). To determine whether this high H+ permeability could be explained by the presence of lipid contaminants such as weak acids, we investigated the effects of (a) bovine serum albumin, which can remove some amphiphilic molecules such as free fatty acids, (b) 6-ketocholestanol, which increases the membrane dipole potential, (c) oleic acid, and (d) chlorodecane, which increases the dielectric constant of the lipid bilayer. Our results show that free fatty acids are inefficient protonophores, as compared with carbonylcyanide-m-chlorphenythydrazone, and that the hypothesis of a weak acid mechanism is not valid with glycolipid bilayers. In the presence of deuterium oxide the H+ conductane was reduced significantly, indicating that proton transport through the glycolipid matrix could occur directly by a hydrogen bond process. The passive transport of H+ through the glycolipid matrix is discussed with regard to the activity of the thylakoid ATP synthase and the inner-envelope H(+)-ATPase.     

The effect of dolichol on the permeability properties of phosphatidylcholine bilayers

The permeability of liposomes to water, glucose, Ca2+ and alkaline cations was monitored by recording the change in absorbance at 450 nm using a rapid reaction stopped-flow spectrophotometer. Liposomes were prepared with egg phosphatidylcholine and concentrations of dolichol ranging from 0.1% to 9% (w/w). Net permeability of phosphatidylcholine bilayers to alkaline cations was induced by the incorporation of dolichol. This effect was not observed in the case of non-charged solutes like glucose or in that of alkaline earth cations such as calcium. Permeation of K+ was significantly increased above the phase transition temperature. These results suggest that dolichols may play a role in biological membranes, besides the well-known glycosyl carrier function in the biosynthesis of glycoproteins.