Permeability and morphology of low temperature phases in bilayers of single and of mixtures of phosphatidylcholines

The properties of subtransitions were studied in aqueous dispersions of saturated phosphatidylcholines (PC) by means of permeability measurements, freeze-fracture electron microscopy, and differential scanning calorimetry (DSC). For dispersions of C16PC, a C16PC analog (2,3-dipalmitoyl-cyclopentano-1-phosphocholine with four methylene residues between the nitrogen and the phosphorus atoms) and C17PC, there was good agreement between phase properties (including subtransitions) as observed by DSC and temperature-related permeability. C16PC and C17PC dispersions also displayed a 'crinkled' surface morphology in the subgel state. The phase diagram for mixtures of C14PC and C16PC was consistent with ideal mixing of these two components in the subgel state and also illustrated the relative independence of the subtransition on acyl chain length as compared to the pre- and main transitions. Together, these results indicate that (i) permeability, DSC and freeze-fracture electron microscopy measurements do correlate reasonably well with the existence of a subgel state, (ii) mixtures of lipids with similar acyl chain lengths can be used to investigate subtransitions, (iii) the development of a subtransition appears to be mainly a function of the non-acyl chain moiety of the phospholipid.     

Cholesterol sulfate and Ca(2+) modulate the mixing properties of lipids in stratum corneum model mixtures

The influence of cholesterol sulfate (CS) and calcium on the phase behavior of lipid mixtures mimicking the stratum corneum (SC) lipids was examined using vibrational spectroscopy. Raman microspectrocopy showed that equimolar mixtures of ceramide, palmitic acid, and cholesterol underwent a phase transition in which, at low temperatures, lipids formed mainly a mosaic of microcrystalline phase-separated domains, and above 45 degrees C, a more fluid and disordered phase in which the three lipid species were more miscible. In the presence of Ca(2+), there was the formation of fatty acid-Ca(2+) complexes that led to domains stable on heating. Consequently, these lipid mixtures remained heterogeneous, and the fatty acid molecules were not extensively involved in the formation of the fluid lipid phase, which included mainly ceramide and cholesterol. However, the presence of CS displaced the association site of Ca(2+) ions and inhibited the formation of domains formed by the fatty acid molecules complexed with Ca(2+) ions. This work reveals that CS and Ca(2+) modulate the lipid mixing properties and the lipid order in SC lipid models. The balance in the equilibria involving Ca(2+), CS, and fatty acids is proposed to have an impact on the organization and the function of the epidermis.     

Membrane contact, fusion, and hexagonal (HII) transitions in phosphatidylethanolamine liposomes

The behavior of phosphatidylethanolamine (PE) liposomes has been studied as a function of temperature, pH, ionic strength, lipid concentration, liposome size, and divalent cation concentration by differential scanning calorimetry (DSC), by light scattering, by assays measuring liposomal lipid mixing, contents mixing, and contents leakage, and by a new fluorometric assay for hexagonal (HII) transitions. Liposomes were either small or large unilamellar, or multilamellar. Stable (impermeable, nonaggregating) liposomes of egg PE (EPE) could be formed in isotonic saline (NaCl) only at high pH (greater than 8) or at lower pH in the presence of low ionic strength saline (less than 50 mOsm). Bilayer to hexagonal (HII) phase transitions and gel to liquid-crystalline transitions of centrifuged multilamellar liposomes were both detectable by DSC only at pH 7.4 and below. The HII transition temperature increased, and the transition enthalpy decreased, as the pH was raised above 7.4, and it disappeared above pH 8.3 where PE is sufficiently negatively charged. HII transitions could be detected at high pH following the addition of Ca2+ or Mg2+. No changes in light scattering and no lipid mixing, mixing of contents, or leakage of contents were noted for EPE liposomes under nonaggregating conditions (pH 9.2 and 100 mM Na+ or pH 7.4 and 5 mM Na+) as the temperature was raised through the HII transition region. However, when aggregation of the liposomes was induced by addition of Ca2+ or Mg2+, or by increasing [Na+], it produced sharp increases in light scattering and in leakage of contents and also changes in fluorescent probe behavior in the region of the HII transition temperature (TH). Lipid mixing and contents mixing were also observed below TH under conditions where liposomes were induced to aggregate, but without any appreciable leakage of contents. We conclude that HII transitions do not occur in liposomes under conditions where intermembrane contacts do not take place. Moreover, fusion of PE liposomes at a temperature below TH can be triggered by H+, Na+, Ca2+, or Mg2+ or by centrifugation under conditions that induce membrane contact. There was no evidence for the participation of HII transitions in these fusion events.     

Differential scanning calorimetric study of a homologous series of fully hydrated saturated mixed-chain C(X):C(X + 6) phosphatidylcholines

The successive high-resolution differential scanning calorimetric (DSC) thermograms for aqueous dispersions of a homologous series of mixed-chain phosphatidylcholines, C(X):C(X + 6)PC, have been recorded and analyzed. In this series of saturated mixed-chain phosphatidylcholines, the total number of carbon atoms in the sn-1 acyl chain increases from 11 to 20, and the sn-2 acyl chain is always 6 methylene units longer than the sn-1 acyl chain. In the initial heating DSC thermograms, two prominent endothermic transitions are detected for all the samples prepared from the various C(X):C(X + 6)PCs except C(12):C(18)PC. In contrast, a single exothermic transition is observed on cooling for all the samples except C(13):C(19)PC. The temperature difference between the two endothermic transitions increases linearly as the acyl chain length of C(X):C(X + 6)PC becomes progressively longer. Interestingly, the main phase transition occurs before the subtransition for C(11):C(17)PC dispersions. Our DSC data further demonstrate that the thermodynamic parameters (Tm, delta H, and delta S) associated with the main phase transition for fully hydrated C(13):C(19)PC and other identical MW phosphatidylcholines are inversely related to the corresponding values of the chain-length inequivalence (delta C/CL) for these lipids. This linear relationship can be employed to map the Tm values for aqueous dispersions prepared from a large number of mixed-chain phosphatidylcholines whose values of delta C/CL are within the range of 0.1-0.4.     

Calorimetric and spectroscopic studies of the phase behavior and organization of lipid bilayer model membranes composed of binary mixtures of dimyristoylphosphatidylcholine and dimyristoylphosphatidylglycerol

The thermotropic phase behavior of hydrated bilayers derived from binary mixtures of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylglycerol (DMPG) was investigated by differential scanning calorimetry, Fourier-transform infrared spectroscopy and 31P-nuclear magnetic resonance spectroscopy. Binary mixtures of DMPC and DMPG that have not been annealed at low temperatures exhibit broad, weakly energetic pretransitions (approximately 11-15 degrees C) and highly cooperative, strongly energetic gel/liquid-crystalline phase transitions (approximately 23-25 degrees C). After low temperature incubation, these mixtures also exhibit a thermotropic transition form a lamellar-crystalline to a lamellar gel phase at temperatures below the onset of the gel/liquid-crystalline phase transition. The midpoint temperatures of the pretransitions and gel/liquid-crystalline phase transitions of these lipid mixtures are both maximal in mixtures containing approximately 30 mol% DMPG but the widths and enthalpies of the same thermotropic events exhibit no discernable composition dependence. In contrast, thermotropic transitions involving the Lc phase exhibit a very strong composition dependence, and the midpoint temperatures and transition enthalpies are both maximal with mixtures containing equimolar amounts of the two lipids. Our spectroscopic studies indicate that the Lc phases formed are structurally similar as regards their modes of hydrocarbon chain packing, interfacial hydration and hydrogen-bonding interactions, as well as the range and amplitudes of the reorientational motions of their phosphate headgroups. Our results indicate that although DMPC and DMPG are highly miscible, their mixtures do not exhibit ideal mixing. We attribute the non-ideality in their mixing behavior to the formation of preferential PC/PG contacts in the Lc phase due to the combined effects of steric crowding of the DMPC headgroups and charge repulsion between the negatively charged DMPG molecules.     

Effect of lipid composition upon fusion of liposomes with Sendai virus membranes

How the lipid composition of liposomes determines their ability to fuse with Sendai virus membranes was tested. Liposomes were made of compositions designed to test postulated mechanisms of membrane fusion that require specific lipids. Fusion does not require the presence of lipids that can form micelles such as gangliosides or lipids that can undergo lamellar to hexagonal phase transitions such as phosphatidylethanolamine (PE), nor is a phosphatidylinositol (PI) to phosphatidic acid (PA) conversion required, since fusion occurs with liposomes containing phosphatidylcholine (PC) and any one of many different negatively charged lipids such as gangliosides, phosphatidylserine (PS), phosphatidylglycerol, dicetyl phosphate, PI, or PA. A negatively charged lipid is required since fusion does not occur with neutral liposomes containing PC and a neutral lipid such as globoside, sphingomyelin, or PE. Fusion of Sendai virus membranes with liposomes that contain PC and PS does not require Ca2+, so an anhydrous complex with Ca2+ or a Ca2+-induced lateral phase separation is not required although the possibility remains that viral binding causes a lateral phase separation. Sendai virus membranes can fuse with liposomes containing only PS, so a packing defect between domains of two different lipids is not required. The concentration of PS required for fusion to occur is approximately 10-fold higher than that required for ganglioside GD1a, which has been shown to act as a Sendai virus receptor. When cholesterol is added as a third lipid to liposomes containing PC and GD1a, the amount of fusion decreases if the GD1a concentration is low.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermodynamics of mixing of phosphatidylserine/phosphatidylcholine from measurements of high-affinity calcium binding

A simple model system is described that allows measurement of equilibrium Ca2+ binding to multilamellar vesicle mixtures of palmitoyloleoylphosphatidylserine (P,O-PS) and dimyristoleoylphosphatidylcholine (MO,MO-PC). The constraint of the chemical equilibrium among aqueous Ca2+, hydrated P,O-PS/MO,MO-PC, and Ca(PS)2, together with measurements of the Ca2+ concentration in equilibrium with defined PS/PC ratios, enables the determination of the thermodynamic activity of the lipids. The activity coefficient of dilute P,O-PS in PC is analyzed in terms of the partial molal free energy to transfer P,O-PS from an environment of PS to an environment of PC. This study of P,O-PS/MO,MO-PC, by comparison with the earlier study of P,O-PS/P,O-PC [Feigensen, G.W. (1989) Biochemistry 20, 1270-1278], reveals that the excess partial molal free energy to transfer P,O-PS from P,O-PS to P,O-PC is -0.7 kcal mol-1. This free energy change arises in part from the favorable transfer of the negatively charged phosphoserine headgroup from an environment of negative charges to an environment of zwitterions. The contribution of acyl chain mismatch to the partial molal free energy to transfer P,O-PS from P,O-PS to MO,MO-PC is found to be approximately +0.7 kcal mol-1. This value is much larger than that of the excess partial molal free energy of mixing in isotropic solutions of linear hydrocarbons that differ in chain length or unsaturation.     

Lipid miscibility and size increase of vesicles composed of two phosphatidylcholines

The size increase of small unilamellar vesicles composed of binary mixtures either of saturated fatty acid phosphatidylcholines with different chain lengths or of saturated and unsaturated phosphatidylcholines was found to depend on the miscibility properties of the lipid components. No size increase was detected in vesicles formed by two miscible phosphatidylcholines. In vesicles composed of two lipids which are partially immiscible in the gel state, a size increase was observed at temperatures which mainly overlapped the range of temperatures of the lipid phase transition. The rate of size increase of vesicles composed of two lipids which are immiscible in the gel state was faster than that of vesicles composed of two partially immiscible phosphatidylcholines, and the process occurred not only at the temperature ranges of the lipid phase transition, but also when both lipids were in the gel state. The vesicle size increase process occurred without the mixing of the internal content of the vesicles. A model is proposed in which the presence of 'fractures' between membrane regions of different fluidity and/or lipid composition controls the rate of this process.     

Cholesterol-multilipid interactions in bilayers

To extend our knowledge of model membrane systems based upon one lipid component, multi-lamellar bilayers were made of cholesterol with two phospholipids in equimolar ratio, and the enthalpy change delta H of the main phase transition of the temary mixture was measured by differential scanning calorimetry (DSC) as a function of increasing cholesterol concentration c. The lipids were saturated phosphatidylcholines CnPC of acyl chain length n, and as the n of the two lipids became more different (from C13PC/C14PC to C14PC/C15PC to C14PC/C18PC to C14PC/C19PC) distinct breaks in the delta H versus c plots were observed. These mixtures displayed only one broad DSC endotherm. Mixtures of an unsaturated lipid C18: 1PC (dioleoyl) with C16PC or with C18PC showed two peaks, with each peak being associated with its parent lipid. However, the delta H versus c plots for each of these peaks showed an initial independence of cholesterol concentration followed by a dependence on cholesterol concentration. These results indicate that, in lipid mixtures, the type of interaction of cholesterol with each lipid component depends on the concentration of cholesterol present.     

Modifications of lipid structure and their influence on mesomorphism in model membranes: the influence of hydrocarbon chains

The influence of hydrocarbon chains on the temperature (TG-LC) of the gel to liquid-crystalline phase transition of model membranes has been investigated over an extensive variety of phosphatidylcholines (PC). The TG-LC is dependent upon the length of the hydrocarbon chains, on whether or not the chains are saturated or have been modified in some way, and on the position of any modification along the chain. For PC having two different acyl chains (heteroacid PC) in the sn-1 and sn-2 positions, the TG-LC is dependent on the chain position and on the inequivalence of chain penetration into the bilayer. Positional isomers of PC have different TG-LC. The first two double bonds introduced in each chain of a PC cause a much greater reduction in TG-LC and in the enthalpy change of the transition than does the subsequent introduction of additional double bonds. Dipolyunsaturated PC have uncooperative (broad) transitions that occur at low temperatures and have small enthalpy changes. While each PC has unique transitional characteristics, there are a number of patterns in the TG-LC which emerge on consideration of all the available data. One such pattern may be useful in predicting TG-LC from analytical data on the composition and positions of acyl chains of various lipids.     

Detection of phase separation in fluid phosphatidylserine/phosphatidylcholine mixtures.

The nonideal mixing of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine, (16:0, 18:1)PS, and 1,2-didodecenoyl-sn-glycero-3-phosphocholine, (12:1, 12:1)PC, in fluid lamellar model membranes was studied by measuring binding of aqueous Ca2+ ions and by x-ray diffraction. A region of two-phase coexistence was found by invariance of the aqueous concentration and by the appearance of two sets of lamellar spacings. The phases were identified as fluid from the diffuse x-ray diffraction in the wide-angle region. The width of the two-phase coexistence region was greater at higher ionic strength. In 800 mM KCl, the phase boundaries were at PS mole fraction 0.5 and 0.8. In 100 mM KCl, the phase boundaries were at PS mole fraction 0.52 and 0.62. Monte Carlo simulations of the lateral distributions of these PS/PC mixtures show pronounced clustering of the lipids.     

Calorimetric and spectroscopic studies of the phase behavior and organization of lipid bilayer model membranes composed of binary mixtures of dimyristoylphosphatidylcholine and dimyristoylphosphatidylglycerol

The thermotropic phase behavior of hydrated bilayers derived from binary mixtures of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylglycerol (DMPG) was investigated by differential scanning calorimetry, Fourier-transform infrared spectroscopy and ³¹P-nuclear magnetic resonance spectroscopy. Binary mixtures of DMPC and DMPG that have not been annealed at low temperatures exhibit broad, weakly energetic pretransitions (∼11-15 °C) and highly cooperative, strongly energetic gel/liquid-crystalline phase transitions (∼23-25 °C). After low temperature incubation, these mixtures also exhibit a thermotropic transition form a lamellar-crystalline to a lamellar gel phase at temperatures below the onset of the gel/liquid-crystalline phase transition. The midpoint temperatures of the pretransitions and gel/liquid-crystalline phase transitions of these lipid mixtures are both maximal in mixtures containing ∼30 mol% DMPG but the widths and enthalpies of the same thermotropic events exhibit no discernable composition dependence. In contrast, thermotropic transitions involving the L_c phase exhibit a very strong composition dependence, and the midpoint temperatures and transition enthalpies are both maximal with mixtures containing equimolar amounts of the two lipids. Our spectroscopic studies indicate that the L_c phases formed are structurally similar as regards their modes of hydrocarbon chain packing, interfacial hydration and hydrogen-bonding interactions, as well as the range and amplitudes of the reorientational motions of their phosphate headgroups. Our results indicate that although DMPC and DMPG are highly miscible, their mixtures do not exhibit ideal mixing. We attribute the non-ideality in their mixing behavior to the formation of preferential PC/PG contacts in the L_c phase due to the combined effects of steric crowding of the DMPC headgroups and charge repulsion between the negatively charged DMPG molecules.     

Lipid selectivity of the calcium and magnesium ion dependent adenosinetriphosphatase, studied with fluorescence quenching by a brominated phospholipid

1,2-Bis(9,10-dibromooleoyl)phosphatidylcholine (BRPC) has been prepared from dioleoylphosphatidylcholine (DOPC). It is shown that the gel to liquid-crystalline phase transition for BRPC occurs below ca. 5 degrees C and that the motional properties of bilayers of BRPC and DOPC as detected by spin-labeled fatty acids are similar. The ATPase activities of the (Ca2+-Mg2+)-ATPase from rabbit muscle sarcoplasmic reticulum reconstituted with BRPC and DOPC are similar. The brominated lipid quenches the fluorescence of the ATPase and can be used to determine selectivity of lipid binding to the ATPase. We show that there is little selectivity on the basis of fatty acyl chain length. Binding constants for phosphatidylcholines and phosphatidylserines are similar in the absence of calcium, although that for phosphatidylserine decreases in the presence of calcium. Phosphatidylethanolamines binds less strongly than phosphatidylcholines, although the difference is small. The largest difference in binding constants is seen between phosphatidylcholines in the gel and liquid-crystalline phases, with a distribution coefficient of 30 in favor of the liquid-crystalline phase. It is shown that the distribution of the ATPase in mixtures of dipalmitoylphosphatidylcholine and BRPC can be understood in terms of the phase diagram for this mixture of lipids. Activities of the ATPase in the presence of mixtures of lipids can be explained in terms of the relative binding constants obtained from the fluorescence experiments.     

Activating and deactivating roles of lipid bilayers on the Ca(2+)-ATPase/phospholamban complex

The physicochemical properties of the lipid bilayer shape the structure and topology of membrane proteins and regulate their biological function. Here, we investigated the functional effects of various lipid bilayer compositions on the sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA) in the presence and absence of its endogenous regulator, phospholamban (PLN). In the cardiac muscle, SERCA hydrolyzes one ATP molecule to translocate two Ca(2+) ions into the SR membrane per enzymatic cycle. Unphosphorylated PLN reduces SERCA's affinity for Ca(2+) and affects the enzymatic turnover. We varied bilayer thickness, headgroup, and fluidity and found that both the maximal velocity (V(max)) of the enzyme and its apparent affinity for Ca(2+) (K(Ca)) are strongly affected. Our results show that (a) SERCA's V(max) has a biphasic dependence on bilayer thickness, reaching maximum activity with 22-carbon lipid chain length, (b) phosphatidylethanolamine (PE) and phosphatidylserine (PS) increase Ca(2+) affinity, and (c) monounsaturated lipids afford higher SERCA V(max) and Ca(2+) affinity than diunsaturated lipids. The presence of PLN removes the activating effect of PE and shifts SERCA's activity profile, with a maximal activity reached in bilayers with 20-carbon lipid chain length. Our results in synthetic lipid systems compare well with those carried out in native SR lipids. Importantly, we found that specific membrane compositions closely reproduce PLN effects (V(max) and K(Ca)) found in living cells, reconciling an ongoing controversy regarding the regulatory role of PLN on SERCA function. Taken with the physiological changes occurring in the SR membrane composition, these studies underscore a possible allosteric role of the lipid bilayers on the SERCA/PLN complex.     

Dilatometric studies of isobranched phosphatidylcholines

Absolute apparent specific volumes have been obtained for phosphatidylcholine lipids with saturated, isobranched hydrocarbon chains with ni = 15 to 20 carbons, with an emphasis upon phase transition behavior, both equilibrium and kinetic. The temperature of the chain-melting transition extrapolates with increasing chain length to the melting temperature of polyethylene with a small odd/even alternation. There are also odd/even alternations in the volume of transition and in the hysteresis of the chain-melting transition, but with the odd and even reversed when compared with the larger odd/even alternation in the lower solid-solid transition that occurs in the longer chain ni lipids. A phenomenological picture is given for the coalescence of the two transitions for shorter ni lipids and this picture is used to sharpen the discussion of the kinetic mechanism of melting. A temperature-reversal experiment shows that the melting from the lowest temperature crystal or C phase to the fluid F phase does not proceed via the metastable gel G phase for 16i. The dilatometric results are combined with recent X-ray structural results for the C and G phases of 17i and 20i to deduce various structural information, including the hydration numbers and the volume of the headgroup, VH = 341 A3, which agrees very well with VH for straight-chain phosphatidylcholines. For the chain-melted F phase the assumption that the methylene volumes of the different ni lipids should be the same at the same temperature is used to obtain the volumes of the methylene and the methyl groups.     

Characterization of actin- and lipid-binding domains in severin, a Ca(2+)-dependent F-actin fragmenting protein.

Severin is a Ca(2+)-activated actin-binding protein that nucleates actin assembly and severs and caps the fast growing ends of actin filaments. It consists of three highly conserved domains. To investigate the domain structure of severin, we constructed genetically the N-terminal domain 1, the middle domain 2, and the tandem domains 2 + 3. Their interaction with actin, Ca2+, and lipids was characterized. Domain 1 contains the F-actin capping and a Ca(2+)-binding site [Eichinger, L., Noegel, A. A., & Schleicher, M. (1991) J. Cell Biol. 112, 665-676]. Binding of domain 2 to actin filaments was Ca(2+)-dependent and saturated at a 1:1 molar ratio. In the presence of Ca2+, about 1.5 mol of domains 2 + 3 bound per mole of F-actin subunit. Scatchard analysis gave a Kd of 18 microM for the interaction of domain 2 with F-actin subunits and a Kd of 1.6 microM for domains 2 + 3. Low-shear viscometry, electron microscopy, and low-speed sedimentation assays showed that domains 2 + 3 induced bundling of actin filaments. The influence of PIP2 micelles on the different activities of severin was assayed using native severin and N- and C-terminally truncated fragments. Severin contains at least two PIP2-binding sites since the activities of the two nonoverlapping severin fragments domain 1 and domains 2 + 3 were inhibited by PIP2. The specificity of severin-phospholipid interaction was investigated by studying the regulation of native severin by PIP2 and other pure or mixed phospholipids.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interdigitated lipid bilayers of long acyl chain species of cerebroside sulfate. A fatty acid spin label study

The metastable phase behavior of semi-synthetic species of cerebroside sulfate (CBS), with hydroxy and non-hydroxy fatty acids from 16 to 26 carbons in length, was compared in Li+ and K+ using differential scanning calorimetry. The structure of the metastable and various stable phases formed in the presence of these two cations was investigated using a fatty acid spin label, 16-doxylstearate. A number of stable phases with successively higher phase transition temperatures and enthalpies occur in the presence of K+ (see the preceding paper). Li+ prevents formation of the most stable phases with the highest transition temperatures and enthalpies for all species of CBS. However, it does not prevent a transition from the metastable phase to the first stable phase of the longer chain C24 and C26 species. Furthermore, it allows C24:0h-CBS to undergo a similar transition, in contrast to a high K+ concentration, which prevents it. The spin label has anisotropic motion in the metastable gel phase formed by all species of CBS on cooling from the liquid crystalline phase. The spectra resemble those in gel phase phospholipids. The spin label is partially insoluble in the most stable phases formed by all the lipids, including the unsaturated C24:1 species, preventing further elucidation of their structure using this technique. However, the spin label is soluble in the first stable phase formed on cooling by the longer chain C24:0 and C26:0-CBS in Li+ and K+ and by C24:0h-CBS in Li+, and is motionally restricted in this phase. The motional restriction is similar to that observed in the mixed interdigitated bilayers of asymmetric species of phosphatidylcholine and fully interdigitated bilayers formed by symmetric phospholipids. It strongly suggests that the highly asymmetric long chain species of CBS form a mixed interdigitated bilayer in their first stable gel phases while the metastable phase of these and the shorter chain lipids may be partially interdigitated. The metastable phase of C24:1-CBS is more disordered suggesting that it may not be interdigitated at all. Thus the results suggest that (i) the hydroxy fatty acid inhibits but does not prevent formation of a mixed interdigitated bilayer by long chain species of CBS, (ii) an increase in non-hydroxy fatty acid chain length from 24 to 26 carbons promotes it, and (iii) a cis double bond probably prevents any form of interdigitation. These results may be relevant to the physiological and pathological roles of these structural modifications of CBS.     

Purification and characterization of (Na+, K+)-ATPase. V. Conformational changes in the enzyme Transitions between the Na-form and the K-form studied with tryptic digestion as a tool.

1. Purified (Na+, K+)-ATPase consisting of membrane fragments was digested with trypsin. The time course of enzyme inactivation was related to the electrophoretic pattern of native and cleaved proteins remaining in the membrane. 2. Differences in both the inactivation kinetics and the cleavage of the large chain (mol. wt 98 000) allow distinction of two patterns of tryptic digestion of (Na+, K+)-ATPase seen with Na+ or K+ in the medium. 3. With K+, the inactivation of (Na+, K+)-ATPase is linear with time in semilogarithmic plots and the activity is lost in parallel with cleavage of the large chain to fragments with molecular weights 58 000 and 48 000. 4. With Na+, the inactivation curves are biphasic. In the initial phase of rapid inactivation, 50% of the activity is lost with minor changes in the composition of the large chain. In the final phase, the large chain is cleaved at a low rate to a fragment with a molecular weight of 78 000. 5. It is concluded that the regions of the large chain exposed in the presence of K+ are distinct from the regions exposed in presence of Na+ and that two conformations of (Na+, K+)-ATPase can be sensed with trypsin, a (t)K-form and a (t)Na-form. 6. Reaction of the (t)K-form with ATP cause transition to the (t)Na-form. Relatively high concentrations of ATP are required and Mg2+ is not necessary. Phosphorylation of (Na+, K+)-ATPase is accompanied by transition from the (t)Na-form to the (t)K-form. Previous kinetic data suggest that these conformational changes are accompanied by shifts in the affinities of the enzyme for Na+ and K+.     

1,2-Dioleoylglycerol promotes calcium-induced fusion in phospholipid vesicles.

The effect of 1,2-dioleoyglycerol (1,2-DOG) on the promotion of Ca(2+)-induced fusion of phosphatidylserine/phosphatidylcholine (PS/PC) vesicles was studied. 1,2-DOG is able to induce the mixing of membrane lipids at concentrations of 10 mol% without mixing of vesicular contents. At concentrations of 20 mol% or higher, 1,2-DOG promotes fusion, lipid and content mixing, of LUV composed of an equimolar mixture of PS and PC, which otherwise are unable to fuse in the presence of Ca2+. Fusion was demonstrated by fluorescence assays monitoring mixing of aqueous vesicular contents and mixing of membrane lipids. Studies by Fourier transform infrared spectroscopy provided evidence for a fusion mechanism different to that of Ca(2+)-induced fusion of pure PS vesicles. Final equilibrium structures were characterized by 31P-NMR and freeze-fracture electron microscopy. Ca(2+)-induced fusion of 1,2-DOG containing vesicles is accompanied by the formation of isotropic structures which are shown to correspond to structures with lipidic particle morphology. The possible fusion mechanisms and implications are discussed.     

Phosphatidylcholine-cholesterol interactions: bilayers of heteroacid lipids containing linoleate lose calorimetric transitions at low cholesterol concentration

Model membranes composed of cholesterol plus one of two phosphatidylcholines (PC), each containing a saturated and a dienoic acyl chain, have been studied by differential scanning calorimetry. The gel to liquid-crystalline phase transition temperature of 1-palmitoyl-2-linoleoyl PC was -19.5 degrees C and that of 1-stearoyl-2-linoleoyl PC was -13.7 degrees C. The addition of cholesterol to the phosphatidylcholines in aqueous dispersion resulted in the progressive removal of the phase transition as observed by differential scanning calorimetry. Per mole of sterol in the membrane, cholesterol was more effective at reducing the enthalpy change of the phase transitions of these bilayers containing dienoic phosphatidylcholines than it is in eliminating the transition of membranes made with other phospholipids that contain more saturated chains. No transitions in membranes made with palmitoyl-linoleoyl PC or stearoyl-linoleoyl PC could be detected calorimetrically when 17 mol% cholesterol was present.