The activation energy of incorporation of extrinsic probes in model vesicles

Time dependence of fluorescence enhancement of probes after addition to lipid vesicles has been used to investigate the position of chromophores in the lipid bilayer. Incorporation studies of a series of $\text{n-(9-}\text{anthroyloxy}$) fatty acids ($\text{n}\text{= 2, 2, 12}\text{and}\text{16}$) and 1,6-diphenylhexatriene in dipalmitoyl phosphatidylcholine vesicles are described. The activation energies for incorporation of these several lipid-mimic type fluorescent probes have been measured. Results show that the activation energy is a function of the distance of the anthracene moiety (chromophore) from the polar end of the probe and the length of the acyl portion of the probe. An average insertion energy of 0.6 kcal/carbon is seen for these fatty acid probes. The activation energy of 1,6-diphenylhexatriene, a factor of 2 greater than that of 16-(9-anthroyloxy)palmitic acid, is consistent with locating 1,6-diphenyl-hexatriene in the middle of the bilayer.     

Interaction of fluorescence quenchers with the n-(9-anthroyloxy) fatty acid membrane probes

The fluorescence quenching of the $\text{n-(9-}\text{anthroyloxy}\text{)}$ (AO) fatty acid probes has been investigated in aqueous dispersions, vesicles of egg phosphatidylcholine and vesicles formed from red cell ghosts. Negatively charged (KI), neutral (acrylamide) and positively charged (CuSO₄) quenchers were used to monitor the location of the probes. The fluorescence of the probes, with the exception of the shortest chain (11-(9-anthroyloxy)undecanoic acid) is not quenched by acrylamide when associated with vesicles. This indicates that in association with vesicles, the 9-anthroyloxy moiety of the long chain probes is buried within the hydrocarbon region and thus well shielded from the aqueous phase. Measurements with KI indicate that the probes are present in the membrane at depths corresponding to the position of the 9-anthroyloxy moiety on the fatty acid, and that the quencher itself forms a concentration gradient within the membrane. Very little or no CuSO₄ quenching was observed for $\text{n-}\text{(9-anthroyloxy)stearic}$ acid probes $\text{(n-}\text{AS)with}\text{n > 2}$, suggesting that in these vesicles Cu²⁺ does not significantly penetrate the bilayer.     

Structural and dynamical studies of mixed chlorophyll/ phosphatidylcholine bilayers via x-ray diffraction,absorption polarization spectroscopy and nuclear magnetic resonance

The structure and dynamics of phosphatidylcholine bilayers containing chlorophyll were studied by X-ray diffraction and absorption polarization spectroscopy in the form of hydrated orientated multilayers below the thermal phase transition of the lipid chains and by nuclear magnetic resonance in the form of single-wall vesicles above the thermal transition. Our results show that (a) chlorophyll is incorporated into the phosphatidylcholine bilayers with its porphyrin ring located anisotropically in the polar headgroup layer of the membrane and with its phytol chain penetrating in a relatively extended form between the phosphatidylcholine fatty acid chains in the hydrocarbon core of the mixed bilayer membrane and (b) the intramolecular anisotropic rotational dynamics of the host phosphatidylcholine molecules are significantly perturbed upon chlorophyll incorporation into the bilayer at all levels of the phosphatidylcholine structure. These dynamics for the host phosphatidtlcholine fatty acid chains are qualitatively different from that of the incorporated chlorophyll phytol chains on a $\text{10}{}^{\mathrm{?9}}\text{? 10}{}^{\mathrm{?10}}\text{s}$ time scale in the ideally mixed two-component bilayer.     

Effects of free fatty acids as membrane components on permeability of drugs across bilayer lipid membranes. A mechanism for intestinal absorption of acidic drugs

Studies of the influence of fatty acids, which were the component of intestinal mucosal lipids, on the permeability of several drugs across bilayer lipid membranes generated from egg phosphatidylcholine and intestinal lipid have been pursued. The permeability coefficients of $\text{p-}\text{aminobenzoic}$ acid, salicylic acid and $\text{p-}\text{aminosalicylic}$ acid (anionic-charged drug) increased when fatty acids such as lauric, stearic, oleic, linoleic and linolenic acid were incorporated into the bilayer lipid membranes generated from phosphatidylcholine. In the presence of methyl linoleate and oleyl alcohol, no enhancing effect on $\text{p-}\text{aminobenzoic}$ acid transfer was obtained. The effect of fatty acids was more marked at pH 6.5 than at pH 4.5. In contrast, upon the addition of fatty acids to intestinal lipid membranes which originally contained fatty acids, the permeability coefficient of $\text{p-}\text{aminobenzoic}$ acid tended to decrease, though the permeability through intestinal lipid membranes was larger than that of phosphatidylcholine membranes. The permeability of $\text{p-}\text{aminobenzoic}$ acid across bilayer lipid membranes from intestinal phospholipids was significantly decreased to about equal that of phosphatidylcholine membranes, and reverted to the value of intestinal lipid membranes when fatty acids were added to intestinal phospholipids. It seemed reasonable to assume that free fatty acids in the intestinal neutral lipid fraction could contribute to the increase in the permeability of $\text{p-}\text{aminobenzoic}$ acid. On the basis of above results, possible mechanisms for good absorbability of weakly acidic drugs from the intestine are discussed.     

Temperature adaptation of biological membranes. The effects of acclimation temperature on the unsaturation of the main neutral and charged phospholipids in mitochondrial membranes of the carp (cyprinus carpio L.)

The phospholipid composition, fatty acid pattern and cholesterol content are studied in mitochondria of red lateral muscle of carp acclimated to high and low environmental temperatures.The results of the experiments are: mitochondria from cold-acclimated carp contain higher proportions of ethanolamine phosphatides than mitochondria from warm-acclimated fish, the opposite is true for the choline phosphatides. Thus, at constant pH, the membrane phospholipids are slightly more negatively charged at low acclimation temperature. The total plasmalogen content is reduced in the cold; this reduction is caused by a decrease in the proportion of the choline plasmalogens. The ethanolamine phosphoglycerides contain approx. 20% of the alk-1-enyl acyl type, irrespective of the acclimation temperature. There is no temperature-dependent difference in the low proportion of cholesterol.The fatty acids of total mitochondrial phospholipids are characterized by large amounts of the $\text{n-3}\text{and}\text{n-6}$ families. The ratio of unsaturated to saturated fatty acids and the unsaturation index are remarkably higher than those reported for comparable mammalian phospholipids. Cold acclimation of carp does not significantly increase the unsaturation of total phospholipids. A fatty acid analysis of the main isolated phospholipids, however, shows that cold acclimation considerably increases unsaturation of the neutral phosphatidylcholine, whereas it dramatically decreases unsaturation of the negatively charged cardiolipin. It is suggested that the observed fatty acid substitution in phosphatidylcholine indicates a temperature-induced fluidity adaptation within the mitochondrial lipid bilayer, whereas the inverse acclimation pattern of cardiolipin provides a suitable lipid to accommodate the temperature-dependent modifications in the dynamic surface shape of integral membrane proteins.     

Transversal mobility in bilayer membrane vesicles: Use of pyrene lecithin as optical probe

Pyrene lecithin, a new excimer-forming lipid molecule, has been synthesized to examine the transversal mobility of probe molecules in lecithin bilayer vesicles. The rate of the lipid exchange is obtained by following the excimer yield as a function of time after mixing of fluorescence doped and undoped vesicles. A rapid exchange ($\text{τ}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 11}\text{s}$) is followed by a slow transfer ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 8}\text{h}$). Above the lipid phase transition the fast transfer can be attributed to an exchange of lipid molecules from the outer layer of one vesicle to the outer layer of another one. The slow exchange is interpreted in terms of the ‘flip-flop’ process between the two layers of a single bilayer vesicle.Using pyrene and pyrene decanoic acid as probe molecules only the fast transfer through the water phase is observed ($\text{τ}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 4}\text{s}$ for pyrene and $\text{τ}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 7}\text{s}$ for pyrene decanoic acid). This indicates that molecules like fatty acids or apolar membrane constituents must equilibrate very rapidly in a single bilayer vesicle.The water solubility or the critical micelle concentration of the probe molecules is determined and related to the transfer rates. An exchange process through the water phase via a monomeric state can be excluded.     

The molecular organisation of bimolecular lipid membranes. The dielectric structure of the hydrophilic/hydrophobic interface

Improvements to a previously described very low-frequency impedance-measuring technique have now allowed the characterisation of a third, electrically distinct, type of substructural region in phosphatidylcholine bimolecular lipid membranes. This region was found to have properties intermediate to those of the hydrophobic (hydrocarbon) layer and the regions containing the polar heads of the phosphatidylcholine molecules. Its properties are consistent with it being associated with the oxygen-rich carboxyl ester portions of the phosphatidylcholine molecules which lie at the hydrophilic/hydrophobic interface. We will refer to these regions in the membrane as the acetyl regions.The individual properties of the three distinct types of regions in the phosphatidylcholine membranes were determined at KCl electrolyte concentrations of 1, 10, 100 and 1000 mM. It was found that with increasing KCl concentration: (a) The capacitance, $\text{C}{}_{\mathrm{<mtext>H</mtext>}}$, of the hydrophobic region increased slightly, indicating a decrease in the thickness of this region. (b) The conductance, $\text{G}{}_{\mathrm{<mtext>H</mtext>}}$, of this hydrophobic region increased by a factor of 20 in going from 1 to 1000 mM KCl electrolyte. (c) The capacitance of the acetyl region was independent of KCl concentration although its conductance increased 5-fold over the range 1–1000 mM KCl. (d) The volume-specific electrical properties of the region containing the polar heads appeared to be essentially independent of KCl concentration. However, a change in thickness of these regions was observed which was consistent with the cholinephosphate dipole being oriented normal to the bilayer surface in 1 mM KCl and parallel to the surface in 1000 mM KCl external solutions.     

Clustering of fatty acids in phospholipid bilayers

From electrophoresis experiments it is concluded that acidic phospholipids incorporated in liquid crystalline phosphatidylcholine bilayers at neutral pH are randomly distributed. The same is true for spin-labelled fatty acids. In contrast, long chain fatty acids are not fully ionized at neutral pH and appear to be clustered, i.e. they segregate out into patches. Only at $\text{pH}\text{>11}$ is the fatty acid-COOH group fully ionized and charge repulsion leads to a random distribution of the fatty acid within the plane of the bilayer.     

The relationship between plasma membrane lipid composition and physical-chemical properties. I. Fluorescence polarization studies of fatty acid-altered EL4 tumor cell membranes

EL4 cells were cultured with exogenous fatty acids under conditions that resulted in their incorporation into membrane phospholipids. The behavior of the fluorescent lipid probes diphenylhexatriene and perylene was monitored in intact EL4 cells and in isolated EL4 plasma membranes. In whole cells substituted with unsaturated fatty acids, there was always a marked decrease in the $\text{P}$ value of both probes compared to the $\text{P}$ value of the probes in unsubstituted cells. In whole cells substituted with saturated fatty acids, on the other hand, $\text{P}$ values for both probes were unchanged compared to unsubstituted cells. In plasma membrane isolated from EL4 cells, no difference in $\text{P}$ values for either probe was observed among membranes from unsubstituted, saturated fatty acid substituted or unsaturated fatty acid substituted cells, even when the degree of fatty acid substitution was quite substantial. Most of the fluorescent signal for both probes in whole cells appeared to come from cytoplasmic lipid droplets. The value of techniques such as fluorescent polarization for monitoring physical properties of membranes (such as ‘fluidity’) is discussed.     

The thickness of monoolein lipid bilayers as determined from reflectance measurements

Measurements of the reflectance of monoolein $\text{n-}\text{alkane}$ and monoolein/squalene lipid bilayers have been made. The total thickness of the bilayer was calculated from the dependence of reflectance on the refractive index of the aqueous salt or sucrose solution surrounding the bilayer. The total thickness was then compared to the thickness of the hydrocarbon chain region as determined from capacitance measurements. From this comparison, we found that the thickness of each polar region of the bilayers in salt solutions was $\text{0.5 ± 0.1}\text{nm}$, independent of the hydrocarbon solvent used. When the aqueous solutions contained sucrose, each polar region was approx. 0.9 nm thick. When $\text{n-}\text{tetradecane}$ and $\text{n-}\text{hexadecane}$ were used as solvents, microlenses of solvent trapped in the monoolein bilayer increased the reflectance. After about one hour, the coalescence of microlenses into larger lenses allowed the reflectance of the bilayer alone to be measured. The use of reflectance to measure the thickness of monoolein bilayers appears to be consistent with other methods and to give useful information about the structure of lipid bilayers.     

Local measurement of lateral motion in erythrocyte membranes by photobleaching technique

The lateral diffusion coefficients ($\text{D}$) of the molecular fluorescence probe 3,3′-dioctadecylindocarbocyanine iodide (DII) in the membrane of discoid erythrocyte ghosts has been measured with the photobleaching technique between 7°C and 40°C. A fluorescence microscope which allows bleaching experiments within small local fields (approx. 1 μm²) at high magnification (X1600) has been used for these measurements. The diffusion coefficient increases from $\text{D = 9 · 10}{}^{\mathrm{?10}}\text{cm}{}^2\text{/s}$ to $\text{D = 7.5 · 10}{}^{\mathrm{?9}}\text{cm}{}^2\text{/s}$ from 7 to 40°C. An increase in membrane fluidity between 12°C and 17°C indicates a conformational change of the lipid bilayer moiety in this temperature region. The diffusion coefficient measured in the regions between the spicules of echinocytes is appreciably smaller than in the untransformed discoid ghosts. In the myelin tubes originating from cells, the lateral diffusion is somewhat larger (about a factor of 2) than in the non-transformed ghosts. With the fluorescence probe technique the rate of growth of myelin tubes of 0.3 μm diameter has been estimated.     

Lipid-protein interactions in model membranes from bovine brain white matter. An ESR spin label and electron microscopy study

Lipid-protein model membranes, prepared from bovine brain white matter and containing all the lipids and Folch-Lees proteolipids, have been studied in macroscopically oriented multibilayers. To examine the lipid environment the membranes were spin labeled with the cholestane spin label ($\text{3-spiro(2′-(N-}\text{oxyl-4′,4′-dimethyl-oxazolidine))5α-cholestane}$) and a fatty acid spin label ($\text{4′-,4′-dimethyloxazolidine-N-}\text{oxyl}$ derivative of 5-ketostearic acid). The ESR spectra exhibit two components arising from fairly well oriented and completely unoriented lipids. Up to a temperature of 55°C the amount of oriented lipids is almost constant, being about 35%. At higher temperatures this percentage drops rapidly to zero. It is shown that the presence of unoriented lipids arises mainly from disrupted areas in the lipid bilayer structure. This is confirmed by electron microscopy and from an analysis of the temperature dependence of the order parameters of the spin labels. The presence of locally disrupted lipid parts in the bilayer is discussed in relation to the interaction of the brain white matter lipids with Folch-Lees protein.     

Interaction of DDT (1,1,1-trichloro-2,2-BIS(p-chlorophenyl)-ethane with liposomal phospholipids

The influence of $\text{1,1,1-}\text{trichloro}\text{-2,2-}\text{bis}\text{(p-}\text{chlorophenyl}\text{)}\text{ethane}$ (DDT) and several other pesticides on the physical state of membrane phospholipids was investigated using model lipids. The thermal dependence of fluorescence intensity of the probe parinaric acid in dipalmitoylphosphatidylcholine liposomes and lipid vesicles of mixed composition were recorded. DDT was incorporated into the liposomal bilayer. The insecticide lowered the phase transition temperature and broadened the temperature range of the transition. The effects were concentration-dependent.The results may be interpreted as a sort of blurred and facilitated phase transition of bilayer lipids caused by intercalation of DDT between fatty acyl chains of membrane phospholipids.     

The influence of phospholipid polar groups on gramicidin channels

The influence of well-defined changes in the polar part of phospholipid molecules on the properties of black lipid membranes was studied using a series of phospholipids with identical hydrocarbon chains, but systematically changed polar groups. The hydrocarbon tails of the lipids under study were composed of 1,2-dipentadecylmethylidene glycerol. The polar parts differed in the degree of $\text{N-}\text{methylation}$ and comprised phosphocholine, $\text{-N,N-}\text{dimethylethanolamine}$, $\text{-N-}\text{methylethanolamine}$ and ethanolamine. Stable black lipid membranes could be formed with the solvents octane, decane, dodecane, tetradecane and hexadecane. The properties of gramicidin-induced single ionic channels changed systematically in membranes from the phosphatidylcholine to the phosphatidylethanolamine analogue, as indicated by an increase in the amplitude A of the unit conductance step and a decrease in the average channel life-time or duration τ. The series of τ-values was opposite to that expected from hydrocarbon thickness (specific capacitance). It is suggested that the surface tension γ is a relevant parameter for the prediction of τ-values.     

Interaction of fatty acids with lipid bilayers

We present a method by which it is possible to describe the binding of fatty acids to phospholipid bilayers. Binding constants for oleic acid and a number of fatty acids used as spectroscopic probes are deduced from electrophoresis measurements. There is a large shift in $\text{p}\text{K}$ value for the fatty acids on binding to the phospholipid bilayers, consistent with stronger binding of the uncharged form of the fatty acid. For dansylundecanoic acid, fluorescence titrations are consistent with the binding constants derived from the electrophoresis experiments. For 12-(9-anthroyloxy)stearic acid, fluorescence and electrophoresis data are inconsistent, and we attribute this to quenching of fluorescence at high molar ratios of 12-anthroylstearic acid to phospholipid in the bilayer.     

Biochimica et Biophysica Acta: Vol. 307 (1973)

The present study evaluates the unsaturated fatty acid requirement in Escherichia coli. A derivative of a double mutant defective both in unsaturated fatty acid biosynthesis and in fatty acid degradation has been selected which grows equally well on anteisopentadecanoate ($\text{12-Me-14:0}$) or $\text{cis-Δ}{}^9\text{-}\text{octadecenoate}$ ($\text{cis-δ}{}^9\text{-18:1}$). When this strain is grown for many generations on $\text{12-Me-14:0}$, there is extensive incorporation of this analogue into the membrane phospholipid and essentially no detectable unsaturated fatty acids residues in any lipid-containing structures of the cell envelope. Secondly, as the maximal growth temperature of E. coli is approached, the minimum content of unsaturated fatty acid required by this strain for growth decreases to a few percent and is associated with the appearance of substantial amounts of $\text{12:0}$ (8%) and $\text{14:0}$ (50%) in the phospholipid. These experiments demonstrate that the cis unsaturated fatty acids of E. coli phospholipids can be replaced by residues which possess no special electronic configuration. Hence, the unsaturated fatty acids do not participate in specific interactions with other membrane components but serve a general role of controlling the packing of paraffin chains in the membrane bilayer.     

Membrane lipid dynamics and enzymic activity in bovine adrenal cortex microsomes

The lipid dynamics of the adrenocortical microsomal membranes was studied by monitoring the fluorescence anisotropy and excited state lifetime of a set of anthroyloxy fatty acid probes (2-, 7-, 9- and 12-(9-anthroyloxy)-stearic acid (AP) and 16-(9-anthroyloxy)palmitic acid (AS). It was found that a decreasing polarity gradient from the aqueous membrane interface to the membrane interior, was present. This gradient was not modified by the proteins, as evidenced by comparison of complete membranes and derived liposomes, suggesting that the anthroyloxy probes were not in close contact with the proteins. An important change of the value of the mean rotational relaxation time as a function of the position of the anthroyl ring along the acyl chain was evidenced. In the complete membranes, a relatively more fluid medium was evidenced in the C₁₆ as compared to the C₂ region, while the rotational motion appeared to be the most hindered at the C₇–C₉ level. In the derived liposomes, a similar trend was observed but the mobility was higher at all levels. The decrease of the mean rotational relaxation time was more important for 12-AS and 16-AP. Temperature dependence of the mean rotational relaxation time of 2-AS, 12-AS and 16-AP in the complete membranes revealed the existence of a lipid reorganization occurring around 27°C and concerning mainly the C₁₆ region. The extent to which the acyl chain reacted to this perturbation at the C₁₂ level depended on pH. The presence of proteins increased the apparent magnitude of this reorganization and also modified the critical temperature from approx. 23°C in the derived liposomes to approx. 27°C in the complete membranes. Thermal dependence of the maximum velocity of the $\text{3-}\text{oxosteroid}\text{Δ}{}^5\text{?Δ}{}^4\text{-}\text{isomerase}$, the second enzyme in the enzymatic sequence, responsible for the biosynthesis of the $\text{3-}\text{oxo}\text{-Δ}{}^4\text{-}\text{steroids}$ in the adrenal cortex microsomes, was studied. The activation energy of the catalyzed reaction was found to be low and constant ($\text{2–5}\text{kcal}\text{·}\text{mol}{}^{\mathrm{?1}}$) in the temperature range 16–40°C at pH 7.5, 8.5 and 9, corresponding to the minimum, intermediate and maximum rate, respectively. A drastic increase of the activation energy ($\text{20}\text{kcal}\text{·}\text{mol}{}^{\mathrm{?1}}$) was observed at temperature below 16°C at pH 7.5. A correlated change of the $\text{p}\text{K}{}_{\mathrm{<mtext>ES</mtext>}}{}^{\mathrm{<mtext>app</mtext>}}$ as function of temperature was detected; at 36°C $\text{p}\text{K}{}_{\mathrm{<mtext>ES</mtext>}}{}^{\mathrm{<mtext>app</mtext>}}\text{= 8.3}$ while at 13°C the value shifted to 8.7. The pH range of the group ionization was narrower at 13°C. In contrast with the behaviour of the $\text{3β-}\text{hydroxy}\text{-Δ}{}^5\text{-}\text{steroid dehydrogenase}$, the $\text{3-}\text{oxosteroid}\text{Δ}{}^5\text{?Δ}{}^4\text{-}\text{isomerase}$ was apparently unaffected by the lipid reorganization at 27°C. It is suggested that this enzyme possesses a different and more fluid lipid environment than the bulk lipids.     

Conformation and packing properties of phosphatidic acid: The crystal structure of monosodium dimyristoylphosphatidate

The conformation and molecular packing of monosodium 1,2-dimyristoyl-sn-glycerophosphate (DMPA) has been determined by single crystal analysis (R = 0.107). The lipid crystallizes in the space group P2₁ with unit cell dimensions: $\text{a = 5.44, b = 7.95, c = 43.98}\text{A}\text{?}$ and β = 114.2°. The two molecules of the unit cell are related by a two-fold screw axis and pack tail-to-tail in a bilayer structure. The monosodium phosphate group packs with rather a small cross-section (24 Ų) relative to the two hydrocarbon chains. This unbalance in packing cross-section is overcome by an interdigitation of the phosphate head groups of adjacent bilayers and the formation of a single, common phosphate group layer at the bilayer interfaces. The phosphate groups are linked by hydrogen bonds to linear strands which laterally are separated by strands of sodium ions. The conformation of the molecules differs from that of other phospholipids. The glycerol chain is oriented parallel (instead of perpendicular) to the layer surface and the parallel stacking of the hydrocarbon chains is achieved by a bend of the γ-chain (instead of the β-chain). Otherwise the conformation of the glycerol dicarboxyl ester group displays the same preferred features as generally found in glycerophospholipids. The hydrocarbon chains pack according to the triclinic (T_∥) packing mode. The interaction and packing principles of the phosphate head group are discussed in relation to the structural behaviour of phosphatidic acid.