Effects of cholesterol- or 7-ketocholesterol-containing liposomes on colony-forming ability of cultured cells

Experiments with cultured Chinese hamster cells showed that incubation of the cells with (phosphatidylcholine + cholesterol + 7-ketocholesterol)-containing liposomes (4:3:1 by weight) during two hours led to a decrease in the colony-forming ability of cells down to zero, while (phosphatidylcholine + cholesterol)-containing liposomes (1:1 by weight) reduce this parameter by 90%. Furthermore, the cholesterol-containing liposomes (without 7-ketocholesterol) induce a decrease in the number of the maximal-site colonies accompanied by the corresponding increase in the number of the middle-size colonies.     

Effect of amphotericin B on cholesterol-containing liposomes of egg phosphatidylcholine and didocosenoyl phosphatidylcholine. A refinement of the model for the formation of pores by amphotericin B in membranes

(1) Binding and K+-permeability measurements were performed on egg and 22 : 1c/22 : 1c-phosphatidylcholine liposomes with or without cholesterol. (2) Amphotericin B binds specifically to cholesterol in both types of liposome despite the difference in bilayer thickness. (3) Addition of amphotericin B to one side of the cholesterol-containing egg phosphatidylcholine bilayers induces a fast K+ efflux from the outermost compartment of the liposomes. In contrast, the total K+ content of sonicated unilamellar cholesterol-containing egg phosphatidylcholine vesicles is released by amphotericin B. (4) Amphotericin B addition to one side of the cholesterol-containing 22 : 1c/22 : 1c-phosphatidylcholine liposomes does not cause a change in K+ permeability. The presence of amphotericin B on both sides of the bilayer, however, induces an increase in K+ permeability. (5) A model is proposed which accounts for the effect of bilayer thickness on the amphotericin B-induced permeability changes in membranes.     

A kinetic method for measuring functional delivery of amphotericin B by drug delivery systems.

The human toxicity of amphotericin B can be considerably reduced by associating the drug with liposomes of varying lipid compositions. Some lipid compositions are much more effective than others. We show that a simple kinetic fluorescence assay using pyranine as an indirect probe of amphotericin-induced K+ currents may be used to study different liposomal drug delivery systems in vitro. We find that lipid mixtures composed of DMPC/DMPG/amphotericin at a 7:3:1 mole ratio show very slow functional delivery with a preference for ergosterol over cholesterol-containing membrane vesicles. On the other hand, amphotericin delivered from egg phosphatidylcholine liposomes lead to 100-fold increases in K+ leakage at one-fifth the amphotericin concentration of the 7:3:1 system. The egg phosphatidylcholine system as well as micellar amphotericin also show a slight selectivity towards cholesterol-containing vesicles over ergosterol. These results are consistent with previous clinical and in vitro cellular studies and this technique may prove valuable in screening of other delivery systems.     

Effect of liposomes containing cholesterol on adenylate cyclase activity of cultured mammalian fibroblasts.

Liposomes prepared with cholesterol and dipalmitoyl phosphatidylcholine were incubated with a clone of normal rat kidney fibroblast of cells in culture. The cells took up [14C]cholesterol in proportion to the concentration of liposomes in the incubation medium, and the uptake increased with time over the four hours of study. Two cell membrane enzymes, adenylate cyclase and (Na+ + K+)-ATPase, exhibited decreased activity after treatment with cholesterol-containing liposomes. The decrease in adenylate cyclase activity was directly proportional to the uptake of [14C]cholesterol. When a variety of subclones of NRK 5W were examined some were found to respond to cholesterol treatment and some did not. These data are consistent with the view that membrane cholesterol content plays a role in controlling the activity of some plasma membrane enzymes.     

Effect of cholesterol on the uptake and intracellular degradation of liposomes by liver and spleen; a combined biochemical and gamma-ray perturbed angular correlation study

We investigated the effect of cholesterol on the uptake and intracellular degradation of liposomes by rat liver and spleen macrophages. Multilamellar vesicles (MLV) consisting of distearoylphosphatidylcholine/phosphatidylserine (molar ratio 9:1) or distearoylphosphatidylcholine/cholesterol/phosphatidylserine (molar ratio 4:5:1) were labeled with [3H]cholesteryl hexadecyl ether and/or cholesteryl [14C]oleate. After i.v. injection the cholesterol-containing liposomes were eliminated less rapidly from the bloodstream and taken up to a lesser extent by the liver (macrophages) than the cholesterol-free liposomes. Assessment of the 3H/14C ratios in liver and spleen cells revealed that the cholesterol-containing liposomes are substantially more resistant towards intracellular degradation than the cholesterol-free liposomes. These results could be confirmed by measuring the release of 111In from liposomes after uptake by liver and spleen by means of gamma-ray perturbed angular correlation spectroscopy. Experiments with cultured Kupffer cells in monolayer also revealed that incorporation of cholesterol results in a decrease of the uptake and an increase of the intracellular stability of cholesteryl [14C]oleate-labeled liposomes. Finally, incubation of both types of liposomes with lysosomal fractions prepared from rat liver demonstrated a difference in susceptibility to lysosomal degradation: the cholesterol-free vesicles were much more sensitive to lysosomal esterase than the cholesterol-containing liposomes. These results may be relevant to the application of liposomes as a drug carrier system to liver and spleen (macrophages).     

Structural analysis of the protein/lipid complexes associated with pore formation by the bacterial toxin pneumolysin

Pneumolysin, a major virulence factor of the human pathogen Streptococcus pneumoniae, is a soluble protein that disrupts cholesterol-containing membranes of cells by forming ring-shaped oligomers. Magic angle spinning and wideline static (31)P NMR have been used in combination with freeze-fracture electron microscopy to investigate the effect of pneumolysin on fully hydrated model membranes containing cholesterol and phosphatidylcholine and dicetyl phosphate (10:10:1 molar ratio). NMR spectra show that the interaction of pneumolysin with cholesterol-containing liposomes results in the formation of a nonbilayer phospholipid phase and vesicle aggregation. The amount of the nonbilayer phase increases with increasing protein concentration. Freeze-fracture electron microscopy indicates the coexistence of aggregated vesicles and free ring-shaped structures in the presence of pneumolysin. On the basis of their size and analysis of the NMR spectra it is concluded that the rings are pneumolysin oligomers (containing 30-50 monomers) complexed with lipid (each with 840-1400 lipids). The lifetime of the phospholipid in either bilayer-associated complexes or free pneumolysin-lipid complexes is > 15 ms. It is further concluded that the effect of pneumolysin on lipid membranes is a complex combination of pore formation within the bilayer, extraction of lipid into free oligomeric complexes, aggregation and fusion of liposomes, and the destabilization of membranes leading to formation of small vesicles.     

The polyene antibiotic amphotericin B acts as a Ca2+ ionophore in sterol-containing liposomes

Amphotericin B (AmB) was shown to induce a Ca2+ influx across ergosterol- and cholesterol-containing large unilamellar liposomes, by following spectrophotometrically the formation of the Arsenazo III-Ca2+ complex. At equivalent antibiotic concentrations the Ca2+ influx was much more extensive through ergosterol-containing membranes (almost 100% with 1 microM AmB, 160 microM lipid) than through cholesterol-containing membranes (below 0.5 microM the influx of Ca2+ was negligible). In the presence of ergosterol-containing membranes the initial rate of Ca2+ influx had the same linear dependence on the ratio antibiotic/lipid whatever the lipid concentration, which was not the case in cholesterol-containing membranes. These results suggest that the channels responsible for the AmB-induced Ca2+ permeability across cholesterol- and ergosterol-containing liposomes have different structures.     

Effect of liposomes containing cholesterol on adenylate cyclase activity of cultured mammalian fibroblasts

Liposomes prepared with cholesterol and dipalmitoyl phosphatidylcholine were incubated with a clone of normal rat kidney fibroblast of cells in culture. The cells took up [¹⁴C]cholesterol in proportion to the concentration of liposomes in the incubation medium, and the uptake increased with time over the four hours of study. Two cell membrane enzymes, adenylate cyclase and ($\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$, exhibited decreased activity after treatment with cholesterol-containing liposomes. The decrease in adenylate cyclase activity was directly proportional to the uptake of [¹⁴C]cholesterol. When a variety of subclones of NRK 5W were examined some were found to respond to cholesterol treatment and some did not. These data are consistent with the view that membrane cholesterol content plays a role in controlling the activity of some plasma membrane enzymes.     

The effect of 25-hydroxycholesterol on accumulation of intracellular calcium.

Liposomes prepared with 25-hydroxycholesterol and egg phosphatidylcholine (PC) were incubated with bovine arterial smooth muscle cells for 8 h at 37 degrees C. Cells incubated in the absence of liposomes or with liposomes containing cholesterol and PC were used as controls. The results indicated that calcium accumulated in the smooth muscle cells incubated in the presence of 25-hydroxycholesterol containing liposomes in an amount proportional to the time of incubation. The calcium accumulation, as indicated by kinetic analysis, resulted from an increased compartment size. (Ca(2+)+Mg2+)-ATPase exhibited decreased activity after pretreatment with 25-hydroxycholesterol containing liposomes and the increased intracellular calcium content was directly proportional to the decreased (Ca(2+) + Mg2+)-ATPase activity. When lipids in the cell membrane were examined, a failure to change the cholesterol/phospholipids ratio in the membrane was noted. The 25-hydroxycholesterol content in the membrane determined by HPLC did not increase. An increase in sphingomyelin and a decrease in phosphatidylethanolamine and acidic phospholipids in the membrane was noted. We suggest that the accumulation of intracellular calcium comes from both an increase of calcium influx and a decrease of (Ca(2+) + Mg2+)-ATPase activity, which may be the consequence of changes in membrane phospholipid composition.     

Effect of phosphatidylinositol replacement by diacylglycerol on various physical properties of artificial membranes with respect to the role of phosphatidylinositol response

In an attempt to gain insight into the physiological role of phosphatidylinositol turnover enhanced by extracellular stimuli, the physical properties of artificial membranes (egg yolk phosphatidylcholine/bovine brain phosphatidylserine) containing phosphatidylinositol or diacylglycerol were studied by ESR using spin probes and freeze-fracture electron microscopy. Diacylglycerol lost both the ability to form lipid bilayer structures and its susceptibility to calcium ions. Yeast phosphatidylinositol included in dipalmitoylphosphatidylcholine liposomes lowered the phase transition temperature of dipalmitoylphosphatidylcholine and expanded the temperature range of phase transition. However, diacylglycerol at the same concentration did not undergo the effects caused by phosphatidylinositol but the phase transition temperature was slightly raised. Phase separation of phosphatidylserine induced by calcium ions was enhanced when the phosphatidylinositol was replaced by diacylglycerol in phosphatidylcholine/ phosphatidylserine/phosphatidylinositol (3:5:2, by molar ratio) mixtures. The mobility of phosphatidylcholine spin probe was decreased in phosphatidylcholine/ phosphatidylserine/diacylglycerol (3:5:2, by molar ratio) liposomes compared with phosphatidylcholine/phosphatidylserine/phosphatidylinositol (3:5:2, by molar ratio) liposomes. An additional component from protonated stearic acid spin probes was observed in phosphatidylcholine/phosphatidylinositol (8:2, by molar ratio) liposomes at 40°C, whereas the component was not seen in phosphatidylcholine/diacylglycerol (8:2, by molar ratio) liposomes. This may indicate the alteration of surface charge induced by the replacement of phosphatidylinositol by diacylglycerol. Indeed, in the presence of 1 mM Ca²⁺, the additional component was removed by an electrostatic interaction between Ca²⁺ and phosphatidylinositol molecules in phosphatidylcholine/phosphatidylinositol liposomes at 40°C. These results support the hypothesis that the enhanced turnover of phosphatidylinositol may play a triggering role for various cellular responses to exogenous stimuli by altering membrane physical states.     

Phospholipid restoration of microsome membranes damaged by Fe2+--ascorbate-dependent lipid peroxidation

The incubation of microsomes damaged by Fe2+--ascorbate-dependent lipid peroxidation with phosphatidylcholine liposomes and micelles is accompanied by the rate decrease of the reduced cytochrome P450 inactivation in microsome membranes. It indicates the elimination of lipid bilayer injuries. The results of study of the saturation degree, surface charge and size of liposomes and micelles influence on the ability to reconstruct the damaged lipid bilayer are presented.     

Fusion of liposomes induced by a cationic protein from the acrosome granule of abalone spermatozoa

Lysin, a protein of Mr 16 000 from the acrosome granule of the abalone, is responsible for the dissolution of the egg vitelline layer. The primary structure of this cationic protein projects some hydrophobic domains in the secondary structure. Lysin was found to associate nonselectively with phospholipid bilayers and cause a spontaneous release of encapsulated carboxyfluorescein in liposomes. The association of lysin with phosphatidylcholine liposomes suggests that there is a hydrophobic interaction between lysin and lipid bilayers. Binding of lysin to phospholipid resulted in the aggregation of phosphatidylserine-containing liposomes, but aggregation was not observed in neutral phosphatidylcholine liposomes. Resonance energy transfer and dequenching of fluorescent 1-palmitoyl-2-cis-parinaroylphosphatidylcholine were both used to determine the fusogenic activity of lysin in aggregated liposomes. Results from both assays are consistent. Lysin-induced fusion was observed in all the phosphatidylserine-containing liposomes, and the general trend of fusion susceptibility was phosphatidylserine/phosphatidylcholine (1:2) approximately equal to phosphatidylserine/phosphatidylcholine/phosphatidylethanolamine (1:1:1) greater than phosphatidylserine/phosphatidylethanolamine (1:2). Cholesterol up to 30% did not affect the intrinsic fusion susceptibility. A hydrophobic penetration by protein molecules and the packing of phospholipid bilayers are used to interpret the fusion susceptibility. Lysin-induced liposome aggregation was highly independent of the state of self-association of lysin in ionic medium. However, the fusogenic activity of self-associated lysin was found to be much less than the monodispersed one. Liposomes preincubated with Ca2+ did not fuse initially as readily as those without Ca2+ treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of phosphatidylinositol replacement by diacylglycerol on various physical properties of artificial membranes with respect to the role of phosphatidylinositol response

In an attempt to gain insight into the physiological role of phosphatidylinositol turnover enhanced by extracellular stimuli, the physical properties of artificial membranes (egg yolk phosphatidylcholine/bovine brain phosphatidylserine) containing phosphatidylinositol or diacylglycerol were studied by ESR using spin probes and freeze-fracture electron microscopy. Diacylglycerol lost both the ability to form lipid bilayer structures and its susceptibility to calcium ions. Yeast phosphatidylinositol included in dipalmitoylphosphatidylcholine liposomes lowered the phase transition temperature of dipalmitoylphosphatidylcholine and expanded the temperature range of phase transition. However, diacylglycerol at the same concentration did not undergo the effects caused by phosphatidylinositol but the phase transition temperature was slightly raised. Phase separation of phosphatidylserine induced by calcium ions was enhanced when the phosphatidylinositol was replaced by diacylglycerol in phosphatidylcholine/phosphatidylserine/phosphatidylinositol (3:5:2, by molar ratio) mixtures. The mobility of phosphatidylcholine spin probe was decreased in phosphatidylcholine/phosphatidylserine/diacylglycerol (3:5:2, by molar ratio) liposomes compared with phosphatidylcholine/phosphatidylserine/phosphatidylinositol (3:5:2, by molar ratio) liposomes. An additional component from protonated stearic acid spin probes was observed in phosphatidylcholine/phosphatidylinositol (8:2, by molar ratio) liposomes at 40 degrees C, whereas the component was not seen in phosphatidylcholine/diacylglycerol (8:2, by molar ratio) liposomes. This may indicate the alteration of surface charge induced by the replacement of phosphatidylinositol by diacylglycerol. Indeed, in the presence of 1 mM Ca2+, the additional component was removed by an electrostatic interaction between Ca2+ and phosphatidylinositol molecules in phosphatidylcholine/phosphatidylinositol liposomes at 40 degrees C. These results support the hypothesis that the enhanced turnover of phosphatidylinositol may play a triggering role for various cellular responses to exogenous stimuli by altering membrane physical states.     

Liposome‐mediated DNA transfer to chicken sperm cells

Abstract

The efficacy of using liposomes to transfer DNA to chicken sperm cells was investigated. Liposomes were prepared from dilauroyl (12:0) phosphatidylcholine (DLPC), dimyristoyl (14:0) phosphatidyl choline (DMPC), dipalmitoyl (16:0) phosphatidylcholine (DPPC), egg yolk phosphatidylcholine (EYPC) or lipids extracted from sperm cell membranes. The efficiency of trapping of DNA into the liposomes, transfer of the DNA from the liposomes to the sperm cells and the effect of the liposomes on the fertilizing ability of the sperm cells were determined. Increasing the concentration of lipid in the liposome preparations increased the trapping efficiency of DNA into liposomes but lowered the transfer of DNA to sperm. Including stearylamine (SA) in the liposomes increased the incorporation of DNA into the liposomes and the DNA transfer to sperm cells, while including lauroyllysophosphatidylcholine (LPC) along with SA resulted in the highest transfer efficiency from liposomes to sperm. The transfer of DNA from liposomes to sperm cells was lowered by increasing the number of sperm cells, while decreasing the number of sperm cells lowered the fertility. The sperm cells remained fertile after exposure to low levels of DPPC or lipofectin reagent or to high levels of SA and LPC. The best conditions for liposome‐mediated gene transfer to chicken sperm cells are thus using either lipofectin reagent at .006 to .06 μmol/ml and 5 × 10⁷ sperm or with DPPC liposomes comprised of 10 μmol/ml total lipid including 5 mol% SA and 20 mol% LPC with 2.5 × 10⁸ sperm cells. The use of liposomes to enhance the transfer of DNA to sperm cells may make the use of sperm cells as gene transfer vectors possible.     

Interaction of cationic liposomes and their DNA complexes with monocytic leukemia cells

Cationic liposomes complexed with DNA have been used extensively as non-viral vectors for the intracellular delivery of reporter or therapeutic genes in culture and in vivo. We examined the relationship between the characteristics of the lipoplexes, their mode of interaction with monocytic THP-1 cells and their ability to transfect these cells. We determined the size and zeta potential of cationic liposomes (composed of 1,2-dioleoyl-3-(trimethylammonium) propane (DOTAP) and its mixtures with neutral lipids), and lipoplexes at different (+/-) charge ratios. As the (+/-) charge ratio of the lipoplexes decreased to (1/1), a significant reduction in zeta potential and an increase in size was observed. The increase in size resulted from fusion between liposomes promoted by DNA, as demonstrated by a lipid mixing assay, and from aggregation of the complexes. Interaction of liposomes and lipoplexes with THP-1 cells was assessed by monitoring lipid mixing ('fusion') as well as binding and cell association. While no lipid mixing was observed with the 1/2 (+/-) lipid/DNA complexes, lipoplexes with higher (+/-) charge ratios underwent significant fusion in conjunction with extensive cell binding. Liposome binding to cells was dependent on the positive charge of the liposomes, and their fusion could be modulated by the co-lipid. DOTAP/phosphatidylethanolamine (1:1) liposomes fused with THP-1 cells, unlike DOTAP/phosphatidylcholine (1:1) liposomes, although both liposome types bound to the cells to a similar extent. The use of inhibitors of endocytosis indicated that fusion of the cationic liposomes with cells occurred mainly at the plasma membrane level. The presence of serum increased the size of the cationic liposomes, but not that of the lipoplexes. Low concentrations of serum (3%) completely inhibited the fusion of cationic liposomes with cells, while inhibiting binding by only 20%. Our results suggest that binding of cationic liposomes and lipoplexes to cells is governed primarily by electrostatic interactions, whereas their fusion is regulated by the lipid composition and sterically favorable interactions with cell surface molecules. In addition our results indicate no correlation between fusion of the lipoplexes with the plasma membrane and the levels of transfection.     

The influence of phospholipid polar head on the lipid hydroperoxide dependent initiation of lipid peroxidation.

In a buffer (Mes) and at a pH (6.5) where Fe2+ is very stable, we have studied the peroxidation of liposomes catalyzed by FeCl2. The liposomes studied, prepared by sonolysis, contained either phosphatidylcholine or 1:1 molar ratio of phosphatidylcholine and phosphatidic acid. The presence of the negatively charged phospholipid causes: 1) rapid Fe2+ oxidation and oxygen consumption; 2) increased generation of lipid hydroperoxides; 3) decreased generation of thiobarbituric acid-reactive materials; 4) very low inhibition of Fe2+ oxidation and lipid hydroperoxide generation by BHT; 5) inhibition of the termination phase of lipid peroxidation at high FeCl2 concentrations. A hypothesis is proposed to explain the results obtained.     

Cholesterol and ergosterol influence nystatin surface aggregation: relation to pore formation

Nystatin interaction with liposomes mimicking fungal and mammalian membranes (ergosterol- and cholesterol-containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) large unilamellar vesicles, respectively) was studied by fluorescence spectroscopy. The activity of this antibiotic was also measured using a pyranine fluorescence detected K+/H+ exchange assay. Nystatin mean fluorescence lifetime varied with the antibiotic concentration and ergosterol content (0-30 mol%) of the lipid vesicles. It sharply increased from 5 to 37 ns upon reaching 100 molecules per liposome, reporting nystatin oligomerization in the membrane. Concomitantly, spectral alterations typical of excitonic coupling were detected and there was a pronounced increase in the initial rate of pore formation by nystatin. These findings suggest that nystatin exerts its antibiotic activity via a two-stage mechanism: at low antibiotic concentrations, surface-adsorbed monomeric antibiotic molecules perturb the lipid packing, changing the permeability properties of the ergosterol-rich liposomes. Upon reaching a critical threshold, nystatin mode of action switches to the classical model of transmembrane aqueous channel formation. In the presence of cholesterol-containing POPC liposomes, neither nystatin spectroscopic properties, nor the kinetics of K+ efflux varied with the antibiotic concentration suggesting that in this case the first stage of antibiotic mode of action always prevails or the assemblies formed by nystatin and cholesterol are very loose.     

Reconstitution of a porcine submaxillary gland beta-D-galactoside alpha 2----3 sialyltransferase into liposomes

Form A of the beta-D-galactoside alpha 2----3 sialyltransferase from porcine submaxillary glands was incorporated into liposomes. Incorporation was achieved by gel filtration of the enzyme in the presence of octylglucoside-phospholipid micelles. As detergent was removed during gel filtration, liposomes (average diameter, 370 A) with bound enzyme were formed and emerged unretarded from the column. The recovery of enzyme activity in the liposomes was about 40% of the initial activity starting with as little as 9 micrograms of transferase. Chromatography on Sepharose CL6B and sucrose density gradient centrifugation confirmed the association of enzyme with liposomes. In contrast to Form A, Form B of the sialyltransferase, which lacks the proposed lipid-binding domain of Form A, cannot be incorporated into liposomes. Form A of the transferase was also incorporated into liposomes composed of phosphatidylcholine, cholesterol, and a mixture of phospholipids from the membranes of the Golgi apparatus from porcine submaxillary glands. Although the transferase was distributed about equally on the internal and external surface of the phosphatidylcholine liposomes, most of the transferase was on the external surface in liposomes containing cholesterol (72%) or in liposomes containing Golgi apparatus phospholipids (88%). The enzyme bound to phosphatidylcholine liposomes was shown by kinetic analysis to have the same activity as that found in the presence of activity-stimulating detergents such as Triton X-100. Enzyme incorporated into cholesterol-containing liposomes had the same activity. In contrast, enzyme bound to liposomes formed from the Golgi apparatus mixed phospholipids had a lower activity, but one similar to that of the transferase in Golgi apparatus membranes. These studies suggest that the composition of a biological membrane may well influence the orientation of the transferase in the membrane as well as modulate its enzymic activity.     

Determination of reflection coefficients of liposomes for some non-electrolytes by osmotic pressure measurement

The reflection coefficients of bilayer lipid vesicles (liposomes) of various compositions have been determined for a number of non-electrolytes. The solutes were the same and the method of measurement was essentially the same as those which have been used to estimate an equivalent pore radius for erythrocytes. The method involves matching the osmotic pressure of solutions of a permeant test solute with that of a known inpermeant solute. Reflection coefficients for cholesterol-containing liposomes and those of erythrocytes are, when account is taken of those solutes known to permeate the erythrocyte by specialized pathways, not greatly different. Lipid bilayers can thus account for most of the permeability characteristics of the cell originally interpreted as due to aqueous pores. Reflection coefficients are significantly higher for egg phosphatidylcholine membranes that contain cholesterol than those which do not. There is a strong correlation between relative permeabilities derived from reflection coefficients and oil-water partition coefficients. There is also good agreement between these permeabilities and permeabilities measured by others, which exhibit an inverse dependence on molecular size. It is suggested that this tendency of membranes to pass small molecules more readily than large molecules, other properties being equal, is a consequence of the surface pressure of the constituent monolayers of the membrane.     

Nanosecond fluorescence anisotropy decays of 1,6-diphenyl-1,3,5-hexatriene in membranes.

Nanosecond decays of the fluorescence anisotropy, r, were studied for the emission of 1,6-diphenyl-1,3,5-hexatriene (DPH) embedded in a series of mixed multilamellar liposomes containing egg yolk phosphatidylcholine, phosphatidylethanolamine and cholesterol in varying molar ratios, as well as in membranes of intact cells and in virus envelopes. The relative contributions of the fast and the infinitely slow decaying component to the steady-state value r, of the fluorescence anisotropy were very similar for artifical and biological membranes. Angles, theta, of the cone, by which the motion of the fluorescent molecule is limited, were calculated from the intensity of the infinitely slow decaying anisotropy component and compared with steady-state fluorescence anisotropies and with 'microviscosities', (eta). An increase in (eta) from 1.5 to 5.2 P in our systems was accompanied by a decrease in theta from 49 degrees to 30 degrees while the decrease in the mean motional relaxation times, phi f, of the label molecule was not more than 1 ns and due mainly to changes in the potential, by which the diffusion of DPH in the membrane is restricted. From these observations we conclude that differences in the steady-state fluorescence anisotropy and in 'microviscosities' of cholesterol-containing membranes (r greater than 0.15) represent changes in the degree of static orientational constraint rather than changes in diffusion rates of the label.