Cardiolipin binds nonyl acridine orange by aggregating the dye at exposed hydrophobic domains on bilayer surfaces.

10-N-Nonyl acridine orange (NAO) has been used at low concentrations as a fluorescent indicator for cardiolipin (CL) in membranes and bilayers. The mechanism of its selective fluorescence in the presence of CL, and not any other phospholipids, is not understood. The dye might recognize CL by its high pK (pK(2)>8.5). To investigate that, we established that NAO does not exhibit a pK in a pH range between 2.3 and 10.0. A second explanation is that the dye aggregates at hydrophobic domains on bilayers exposed by the CL. We found that a similar spectral shift occurs in the absence of CL in a concentrated solution of the dye in methanol and in the solid state. A model is proposed in which the nonyl group inserts in the bilayer at the hydrophobic surface generated by the presence of four chains on the phospholipid.

Do sterols reduce proton and sodium leaks through lipid bilayers?

Proton and/or sodium electrochemical gradients are critical to energy handling at the plasma membranes of all living cells. Sodium gradients are used for animal plasma membranes, all other living organisms use proton gradients. These chemical and electrical gradients are either created by a cation pumping ATPase or are created by photons or redox, used to make ATP. It has been established that both hydrogen and sodium ions leak through lipid bilayers at approximately the same rate at the concentration they occur in living organisms. Although the gradients are achieved by pumping the cations out of the cell, the plasma membrane potential enhances the leakage rate of these cations into the cell because of the orientation of the potential. This review proposes that cells use certain lipids to inhibit cation leakage through the membrane bilayers. It assumes that Na(+) leaks through the bilayer by a defect mechanism. For Na(+) leakage in animal plasma membranes, the evidence suggests that cholesterol is a key inhibitor of Na(+) leakage. Here I put forth a novel mechanism for proton leakage through lipid bilayers. The mechanism assumes water forms protonated and deprotonated clusters in the lipid bilayer. The model suggests how two features of lipid structures may inhibit H(+) leakage. One feature is the fused ring structure of sterols, hopanoids and tetrahymenol which extrude water and therefore clusters from the bilayer. The second feature is lipid structures that crowd the center of the bilayer with hydrocarbon. This can be accomplished either by separating the two monolayers with hydrocarbons such as isoprenes or isopranes in the bilayer's cleavage plane or by branching the lipid chains in the center of the bilayers with hydrocarbon. The natural distribution of lipids that contain these features are examined. Data in the literature shows that plasma membranes exposed to extreme concentrations of cations are particularly rich in the lipids containing the predicted qualities. Prokaryote plasma membranes that reside in extreme acids (acidophiles) contain both hopanoids and iso/anteiso- terminal lipid branching. Plasma membranes that reside in extreme base (alkaliphiles) contain both squalene and iso/anteiso- lipids. The mole fraction of squalene in alkaliphile bilayers increases, as they are cultured at higher pH. In eukaryotes, cation leak inhibition is here attributed to sterols and certain isoprenes, dolichol for lysosomes and peroxysomes, ubiquinone for these in addition to mitochondrion, and plastoquinone for the chloroplast. Phytosterols differ from cholesterol because they contain methyl and ethyl branches on the side chain. The proposal provides a structure-function rationale for distinguishing the structures of the phytosterols as inhibitors of proton leaks from that of cholesterol which is proposed to inhibit leaks of Na(+). The most extensively studied of sterols, cholesterol, occurs only in animal cells where there is a sodium gradient across the plasma membrane. In mammals, nearly 100 proteins participate in cholesterol's biosynthetic and degradation pathway, its regulatory mechanisms and cell-delivery system. Although a fat, cholesterol yields no energy on degradation. Experiments have shown that it reduces Na(+) and K(+) leakage through lipid bilayers to approximately one third of bilayers that lack the sterol. If sterols significantly inhibit cation leakage through the lipids of the plasma membrane, then the general role of all sterols is to save metabolic ATP energy, which is the penalty for cation leaks into the cytosol. The regulation of cholesterol's appearance in the plasma membrane and the evolution of sterols is discussed in light of this proposed role.

Receptor pattern formation as a signal for the capture of lipoproteins.

A critical step in the uptake of dietary cholesterol by the liver is the binding of remnant lipoprotein particles to receptors in the space of Disse. We have found that increases in the cholesterol content of hepactocyte membranes reduces the binding of beta-very low density lipoproteins (beta-VLDL) and decreases internalization. This increase in membrane cholesterol of human hepatoma cells (HepG2) produces a similar effect on binding to primary human fibroblasts. However, receptor-negative familial hypercholesterolemic (FH) fibroblasts lack the ability to respond to membrane cholesterol modification. A polyclonal antibody directed against the C-terminus region of the apo-B,E-(LDL) receptor importantly affects the internalization process, suggesting that protein-protein interactions consolidate the pattern formation of receptors, a process that triggers lipoprotein internalization. We propose that cholesterol interferes with this pattern formation by affecting the lateral movement and organization of the receptors.

Permeation of protons, potassium ions, and small polar molecules through phospholipid bilayers as a function of membrane thickness.

Two mechanisms have been proposed to account for solute permeation of lipid bilayers. Partitioning into the hydrophobic phase of the bilayer, followed by diffusion, is accepted by many for the permeation of water and other small neutral solutes, but transient pores have also been proposed to account for both water and ionic solute permeation. These two mechanisms make distinctively different predictions about the permeability coefficient as a function of bilayer thickness. Whereas the solubility-diffusion mechanism predicts only a modest variation related to bilayer thickness, the pore model predicts an exponential relationship. To test these models, we measured the permeability of phospholipid bilayers to protons, potassium ions, water, urea, and glycerol. Bilayers were prepared as liposomes, and thickness was varied systematically by using unsaturated lipids with chain lengths ranging from 14 to 24 carbon atoms. The permeability coefficient of water and neutral polar solutes displayed a modest dependence on bilayer thickness, with an approximately linear fivefold decrease as the carbon number varied from 14 to 24 atoms. In contrast, the permeability to protons and potassium ions decreased sharply by two orders of magnitude between 14 and 18 carbon atoms, and leveled off, when the chain length was further extended to 24 carbon atoms. The results for water and the neutral permeating solutes are best explained by the solubility-diffusion mechanism. The results for protons and potassium ions in shorter-chain lipids are consistent with the transient pore model, but better fit the theoretical line predicted by the solubility-diffusion model at longer chain lengths.

Water transport across biological membranes.

The rate of the lateral diffusion of straight-chain phospholipids predicts the rate of water diffusion through bilayers. A new model of lipid dynamics integrates these processes. Substances such as cholesterol that reduce water diffusion proportionally reduce lateral diffusion. The model yields a number of predictions about the dynamics of the lipids at the Tm and suggests different mechanisms for how water diffuses across bilayers of other-than-straight-chain lipids, and how proteins bind to membranes. A second recent development in water transport across biological membranes is the discovery of a ubiquitous family of water transport proteins that facilitate large-volume water translocation. Like water diffusion through lipid bilayers, water transport by these proteins is directed by osmosis and is therefore under the control of ATP and ion pumps. The presence of water transport proteins in membranes is often regulated by hormones.

pH-dissociation characteristics of cardiolipin and its 2'-deoxy analogue.

Cardiolipin (CL) is found in inner mitochondrial membranes and the plasma membrane of aerobic prokaryotes. CL is tightly bound to those transmembrane enzymes associated with oxidative phosphorylation. CL has earlier been reported to have a single pK at low pH. We have titrated CL in aqueous suspension (bilayers) and in solution in methanol/water (1:1, vol/vol) and found it to display two different pK values, pK1 at 2.8 and pK2 initially at 7.5 but shifting upwards to 9.5 as the titration proceeds. The unusually high pK2 might be explained by the formation of a unique hydrogen bond in which the free hydroxyl on the central glycerol forms a cyclic intramolecular hydrogen-bonded structure with one protonated phosphate (P-OH group). We have therefore chemically synthesized the 2'-deoxycardiolipin analogue, which lacks the central free hydroxyl group, and measured its pH-dissociation behavior by potentiometric titration, under the same conditions as those for CL. The absence of the hydroxyl group changes the titration dramatically so that the deoxy analogue displays two closely spaced low pK values (pK1 = 1.8; pK2 = 4.0). The anomalous titration behavior of the second dissociation constant of CL may be attributed to the participation of the central glycerol OH group in stabilizing the formation of a cyclic hydrogen-bonded monoprotonated form of CL, which may function as a reservoir of protons at relatively high pH. This function may have an important bearing on proton pumping in biological membranes.

The elasticity of synthetic phospholipid vesicles obtained by photon correlation spectroscopy.

Osmotic-swelling experiments were conducted on a variety of preparations of "uniform" unilamellar vesicle systems. The synthetic lipid preparations included both vesicles produced by extrusion through polycarbonate ultrafiltration membranes and vesicles produced by the pH-adjustment method. The vesicles were monitored by photon correlation spectroscopy during swelling as the osmolarity of the external solution was decreased. Contrary to our previously reported results [Aurora, T. S., Li, W., Cummins, H. Z., & Haines, T. H. (1985) Biochim. Biophys. Acta 820, 250-258; Li, W., & Haines, T. H. (1986) Biochemistry 25, 7477-7483; Li, W., Aurora, T. S., Haines, T. H., & Cummins, H. Z. (1986) Biochemistry 25, 8220-8229; Haines, T. H., Li, W., Green, M., & Cummins, H. Z. (1987) Biochemistry 26, 5439-5447] large unilamellar vesicles produced from acidic lipids by the pH-adjustment technique were highly polydisperse and did not swell in a manner that permitted the computation of a Young's modulus, presumably due to the polydispersity. Also contrary to our previous reports, membranes derived from bovine submitochondrial particles did not produce evidence of swelling when subjected to similar protocols. Analysis of osmotic swelling of extruded unilamellar vesicles has allowed us to assign Young's moduli for bilayers of dioleoylphosphatidylcholine and dioleoylphosphatidylglycerol, in the range (5-8) x 10(8) and (3-6) x 10(8) dyn/cm2, respectively. The diameters and polydispersites obtained with electron microscopy and photon correlation spectroscopy were compared directly and with computer-modeling techniques. While excellent agreement was obtained for distributions with low polydispersity (approximately greater than 0.1), serious disagreement was found when the polydispersity exceeded approximately 0.2.

The elasticity of uniform, unilamellar vesicles of acidic phospholipids during osmotic swelling is dominated by the ionic strength of the media.

Uniform, unilamellar vesicles have been prepared by the pH-modification technique. The initial sizes of the vesicles were from 200 to 700 nm and were measured to within 1-3% by photo correlation spectroscopy. Vesicles were made of the dioleoyl esters of phosphatidic acid, phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, the diphytanyl ethers of phosphatidylglycerol, Escherichia coli lipids, and lac permease reconstituted into E. coli lipids. The vesicle suspensions were prepared and then diluted with electrolyte (KCl) and/or nonelectrolyte (sucrose, trehalose, pentaerythritol) impermeants. The amplitude of the swelling is linearly proportional to the osmotic pressure difference across the bilayer. We have determined the elastic modulus, the elastic limit (percent surface expansion at bursting), and the transbilayer pressure difference at bursting for each of these vesicles at constant osmolarity but at different ionic strengths. We find that the elastic properties of the bilayer vary by a factor of 10 in electrolyte media as compared to isosmolal nonelectrolyte media and that this variation appears to be related to both the charge density at the surface and the ionic strength of the media. Anionic lipid vesicles in 150 mM KCl have a significantly higher modulus (50 X 10(7) dyn/cm2) and transbilayer pressure difference (40 mosM) at bursting with a small capacity to stretch (3-4% surface expansion) compared to the same vesicles suspended in nonelectrolyte impermeants. The latter vesicles undergo a significant surface expansion (8-10%), display a low modulus (3 X 10(7) dyn/cm2), and burst at 3-4 mosM bilayer pressure difference. Vesicles suspended in media of constant osmolarity at various ionic strengths display properties with proportional values.(ABSTRACT TRUNCATED AT 250 WORDS)

Elasticity of synthetic phospholipid vesicles and submitochondrial particles during osmotic swelling.

A rapid and accurate method has been developed for measuring the elastic response of vesicle bilayer membranes to an applied osmotic pressure. The technique of dynamic light scattering is used to measure both the elastic constant and the elastic limit of dioleoylphosphatidic acid (DOPA) and DOPA-cholesterol vesicles and of submitochondrial particles derived from the inner membrane of bovine heart mitochondria. The vesicles prepared by the pH-adjustment method are unilamellar and of uniform size between 240 and 460 nm in diameter. The vesicles swell uniformly upon dilution. The observed change in size is not due to any change in the shape of the vesicles. The data also indicate that the vesicles are spherical and not flaccid. The total vesicle swelling in these studies resulted in a 3-4% increase in surface area for vesicles swollen in 0.15 M KCl and a 5-10% increase in surface area for vesicles swollen in 0.25 M sucrose. This maximum represents the elastic limit of the vesicles. Evidence is presented to show that the vesicles release contents after swelling to this maximum, reseal immediately, and reswell according to the osmotic pressure. For DOPA vesicles in a 0.15 M KCl-tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) buffer (pH 7.55), the observed membrane modulus is found to be in the range of 10(8) dyn/cm2. The modulus was found to be in the order of 10(7) dyn/cm2 for DOPA vesicles in a 0.25 M sucrose-Tris-HCl buffer (pH 7.55). This is comparable to that of submitochondrial particles in the same sucrose-Tris-HCl buffer. The observed membrane modulus also decreases with vesicle size. Its magnitude and its variation with ionic strength indicate that the major component of bilayer elasticity is neither the inherent elasticity of the bilayer nor the bending modulus. The variation of the membrane modulus with respect to curvature suggests that its principal component may be related to surface tension effects including the negative charges on the vesicle surface. There is considerable variation between vesicles swollen in sucrose and those swollen in KCl in the membrane modulus, in the elastic limit at which the vesicles burst, and in the transbilayer pressure difference at bursting. The latter was found to be 4-6 mosM (10(5) dyn/cm2) in sucrose solution and 20-4 mosM (10(6) dyn/cm2) in KCl solution.

Uniform preparations of large unilamellar vesicles containing anionic lipids.

A general procedure for the preparation of large unilamellar vesicles of selected sizes has been developed. The procedure consists of dissolving the lipid in organic solvent, washing with mild acid, removing the solvent, adding salt (0.15 M KCl) solution, and adjusting the pH (raising it to about pH 10 and lowering it immediately to pH 7.55). The procedure takes less than 30 min. The resulting unilamellar vesicles are of a single size with a rather low standard deviation. The sizes of these preparations range between 150 and 1000 nm in diameter. Sizes and polydispersities were measured to within 1-2% by photon correlation spectroscopy. Vesicle size varies with the phospholipid structure, the composition of the phospholipid mixture, the ionic strength of the medium, the alkyl chain composition, the cholesterol content of the phospholipid mixture, and the timing of the pH adjustment procedure. Uniform preparations of vesicles have been obtained from the dioleoyl esters of phosphatidic acid, phosphatidylglycerol, phosphatidylethanolamine, and phosphatidylserine, from diphytanyl ethers of glycolipid sulfate, phosphatidylglycerol, phosphatidylglycerol phosphate, and phosphatidylglycerol sulfate, from bovine liver phosphatidylinositol, from Escherichia coli phosphatidylethanolamine, from membrane lipid extracts from E. coli and Holabacterium cutirubrum, and from dodecanesulfonate-alkanol mixtures and free oleic acid. The preparation of unilamellar vesicles from oleic acid is novel, and the size range is 600-3000 nm; the preparations are relatively uniform. Vesicles of phospholipids in which sucrose and trehalose replace salt as the impermeant do not differ significantly from those prepared in pentaerythritol.

Preparation and characterization of monodisperse unilamellar phospholipid vesicles with selected diameters of from 300 to 600 nm.

A method has been developed for making large unilamellar vesicles (LUV) with low polydispersity. The LUV, constituted of dioleoylphosphatidic acid (DOPA), 300 nm in diameter are made by a modification of the pH adjustment technique (Hauser, H. and Gains, N. (1982) Proc. Natl. Acad. Sci. USA 79, 1683-1687). This size is 10 times that (30 nm) of vesicles prepared by prolonged sonication. Vesicle size is increased stepwise by adding cholesterol (to a maximum of 40 mol% cholesterol) to form vesicles in 0.15 M KCl with up to 600 nm diameter. The vesicle size is measured by photon correlation spectroscopy, electron microscopy, and by measurement of the internal volume with cyanocobalamin while calculating the number of DOPA molecules per vesicle. Vesicles are stable for at least three weeks. Sepharose 4B column chromatography of the preparation yields a peak of fractions with the same polydispersity as the original sample and shows that 30 to 40% of the original lipid in a sample is recovered as LUV. Less than 2% of the sample forms small unilamellar vesicles (SUV) (diameter = 30 nm), which emerge from the column in a separate peak. Since the remaining lipid is not suspended in the buffer during vesicle formation, for most purposes the vesicles may be used immediately after titration so that they can be prepared in less than 40 min.

Anionic lipid headgroups as a proton-conducting pathway along the surface of membranes: a hypothesis.

Evidence has been gathering from several laboratories that protons in proton-pumping membranes move along or within the bilayer rather than exchange with the bulk phase. These experiments are typically conducted on the natural membrane in vivo or in vitro or on fragments of natural membrane. Anionic lipids are present in all proton-pumping membranes. Model studies on the protonation state of the fatty acids of liposomes containing entrapped water show that the bilayers always contain mixtures of protonated and deprotonated carboxylates. Protonated fatty acids form stable acid-anion pairs with deprotonated fatty acids through unusually strong hydrogen bonds. Such acid-anion dimers have a single negative charge, which is shared by the four negative oxygens of both headgroups. The two pK values of the resulting dimer will be significantly different from the pK of the monomeric species, so that the dimer will be stable over a wide pH range. It is proposed that anionic lipid headgroups in biological membranes share protons as acid-anion dimers and that anionic lipids thus trap and conduct protons along the headgroup domain of bilayers that contain such anionic lipids. Protons pumped from the other side of the membrane may enter and move within the headgroup sheet because the protonation rate of negatively charged proton acceptors is 5 orders of magnitude faster than that of water. Protons trapped in the acidic headgroup sheet need not leave this region in order to be utilized by a responsive proton-translocating pore (a transport protein using the proton gradient). Experiments suggest the proton concentration in the headgroup domain may vary widely and the anionic lipid headgroup sheet may therefore function as a proton buffer. Due to the Gouy-Chapman-Stern layer at polyanionic surfaces, anionic lipids will also sequester protons from the bulk solution at low and moderate ionic strengths. At high ionic strength metal cations may replace protons sequestered near the headgroups, but these cations cannot substitute for protons in the "proton-conducting pathway," which is based on hydrogen bonding.

The flagellar membrane of Ochromonas danica. Isolation and electrophoretic analysis of the flagellar membrane, axonemes, and mastigonemes.

The isolation and purification of the flagellar membrane of the phytoflagellate, Ochromonas danica, is described. The procedure is simple, mild, rapid, and it produces a pure membrane preparation. The method additionally permits the isolation of clean preparations of axonemes and mastigonemes from a single flagella preparation. Each component was studied by electron microscopy and acrylamide gel electrophroesis. The isolated flagella preparation was nearly free of other cellular organelles as judged by phase contrast and electron microscopy. The purified membrane preparation consisted of small vesicles (500 to 1500 A in diameter) with a trilamellar pattern about 80 A thick. Isolated membrane was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, displaying five major protein bands, five minor protein bands, and some protein which remained at the origin. The five major protein components had apparent molecular weights of 54,000, 47,000, 35,000, 31,000, and 28,000. All mastigoneme protein components are glycoproteins as judged by periodic acid-Schiff staining. The mastigoneme preparation contained three major protein bands. Two of them were revealed as doublets and migrated with an average velocity corresponding to 83,000 delatons, the other major protein band migrated with a velocity corresponding to 54,000 daltons. A heavy carbohydrate band is seen near the bromphenol blue tracking dye. The axoneme preparation showed one major protein band having an apparent molecular weight of about 54,000 and some proteins having high molecular weights which remained on top of the polyacrylamide gel.

The flagellar membrane of Ochromonas danica. Lipid composition.

The lipids of the whole flagella and the flagella membrane of the phytoflagellate Ochromonas danica were isolated and compared with those of the whole cell. The polar lipids were separated by two-dimensional thin layer chromatography. One-dimensional thin layer chromatography was used for the separation of the nonpolar lipids. In all respects the lipids of the whole flagella were identical with those of the flagellar membrane. These methods established the presence in flagellar membrane of the polychlorosulfolipids of O. danica as more than 90 molar per cent of the total polar lipids. These sulfolipids had been previously characterized as 1,14-docosanediol-1, 14-disulfate and 1,15-tetracosanediol-1,15-disulfate, containing zero to six chloro groups substituting for hydrogen on the chain. Seven unknown polar lipids were found. Both phosphorus analysis on each lipid and the molybdenum spray reagent for phospholipids on the chromatogram showed that there is no phospholipid present in O. danica flagellar membrane. Positive reactions to the diphenylamine spray reagent suggest that up to four of the unknown polar lipids are glycolipids. Of these, three reacted positively with ninhydrin. All of the unknown lipids reacted with the acidified 2,4-dinitrophenylhydrazine spray reagent suggesting the presence of aldehyde, ketone, glycoside, or plasmalogen. One unknown substance appeared near the origin of thin layer chromatograms. It showed a positive reaction with Dragendorff reagent, suggesting the presence of a quaternary amine group. This substance is presumed to be nonlipid, since it is not synthesized from [1-14C]acetate under the growth conditions used, as revealed by autoradiograms of thin layer chromatograms. It contained 35% hexose or hexosamine. It is devoid of phosphorus (0.7%) and is less than 4% protein (or phenolic groups or peptide), as judged by the Lowry assay using bovine serum albumin as a standard. Analysis of the nonpolar lipids of the flagellar membrane showed that free fatty acids constitute about 12 molar per cent of the total lipids. These fatty acids could be true components of the membrane or artifacts of the extraction procedure although every precaution was taken to prevent artifactual production of free fatty acids. The sterols constitute nearly 10 molar per cent of total lipids. Sterol esters were absent from the membrane. There are two additional major unknown nonpolar lipids present. The implications of such a high proportion of chlorosulfolipids as a polar lipid component in the membrane are important because of the unique structures of these lipids, which have ionic groups at or near both ends of the aliphatic chain.