The modulating effects of lipids on purified rat liver Golgi galactosyltransferase

Galactosyltransferase was purified from rat liver Golgi membranes. The Triton X-100, used to solubilize the enzyme was removed immediately prior to the lipid interaction studies. In lipid vesicles, prepared from a variety of phosphatidylcholines (PCs), including egg PC, DOPC, DMPC, DPPC and DSPC, the ability of the lipids to stimulate the enzyme decreased in the order egg PC greater than DOPC greater than DMPC greater than DPPC greater than DSPC, i.e. the lower the transition temperature (Tc) the greater the stimulation of the enzyme. A second, neutral lipid, phosphatidylethanolamine was used to permit a comparison of the effect of a different head group of the same net charge at neutral pH. The PEs included, egg PE, soy PE, Pl-PE, PE(PC) and DPPE in order of increasing Tc. The effect of the PEs was opposite to that of the PCs, i.e. the higher the Tc, the greater the stimulation of the enzyme. In fact egg PE and soy PE which have the lowest Tc values were inhibitory. Thus the modulation of the Golgi membrane galactosyltransferase by these lipids was different from that reported earlier for the bovine milk galactosyltransferase. The effects of two acidic lipids, egg phosphatidic acid (PA) and egg phosphatidylglycerol (PG) were studied also. Both totally inhibited the enzyme even at low concentrations of lipid, however, the PA was more effective than PG. In mixtures of neutral lipid (PC) and acidic lipid (PA or PG), the effect of the acidic lipid dominated. Even in the presence of excess PC, total inhibition of the enzyme was observed. It was concluded that the enzyme bound the acidic lipid preferentially to itself. The choice of the lipids allowed us to make several direct comparisons concerning the effect of the nature of the lipid head group on the activity of the enzyme. For example PE(PC), egg PA and egg PG would have fatty acid chains identical to egg PC since these three lipids are all prepared by modification of egg PC. As well, DPPE differs from DPPC only by nature of the head group. These comparisons indicated that not only the net charge but also chemical nature of the head group were important in the lipid modulation of Golgi galactosyltransferase.     

Effects of lipid composition on membrane permeabilization by sticholysin I and II, two cytolysins of the sea anemone Stichodactyla helianthus

Sticholysin I and II (St I and St II), two basic cytolysins purified from the Caribbean sea anemone Stichodactyla helianthus, efficiently permeabilize lipid vesicles by forming pores in their membranes. A general characteristic of these toxins is their preference for membranes containing sphingomyelin (SM). As a consequence, vesicles formed by equimolar mixtures of SM with phosphatidylcholine (PC) are very good targets for St I and II. To better characterize the lipid dependence of the cytolysin-membrane interaction, we have now evaluated the effect of including different lipids in the composition of the vesicles. We observed that at low doses of either St I or St II vesicles composed of SM and phosphatidic acid (PA) were permeabilized faster and to a higher extent than vesicles of PC and SM. As in the case of PC/SM mixtures, permeabilization was optimal when the molar ratio of PA/SM was ~1. The preference for membranes containing PA was confirmed by inhibition experiments in which the hemolytic activity of St I was diminished by pre-incubation with vesicles of different composition. The inclusion of even small proportions of PA into PC/SM LUVs led to a marked increase in calcein release caused by both St I and St II, reaching maximal effect at ~5 mol % of PA. Inclusion of other negatively charged lipids (phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidylinositol (PI), or cardiolipin (CL)), all at 5 mol %, also elicited an increase in calcein release, the potency being in the order CL approximately PA > PG approximately PI approximately PS. However, some boosting effect was also obtained, including the zwitterionic lipid phosphatidylethanolamine (PE) or even, albeit to a lesser extent, the positively charged lipid stearylamine (SA). This indicated that the effect was not mediated by electrostatic interactions between the cytolysin and the negative surface of the vesicles. In fact, increasing the ionic strength of the medium had only a small inhibitory effect on the interaction, but this was actually larger with uncharged vesicles than with negatively charged vesicles. A study of the fluidity of the different vesicles, probed by the environment-sensitive fluorescent dye diphenylhexatriene (DPH), showed that toxin activity was also not correlated to the average membrane fluidity. It is suggested that the insertion of the toxin channel could imply the formation in the bilayer of a nonlamellar structure, a toroidal lipid pore. In this case, the presence of lipids favoring a nonlamellar phase, in particular PA and CL, strong inducers of negative curvature in the bilayer, could help in the formation of the pore. This possibility is confirmed by the fact that the formation of toxin pores strongly promotes the rate of transbilayer movement of lipid molecules, which indicates local disruption of the lamellar structure.     

Calcium-induced lipid phase separations and interactions of phosphatidylcholine/anionic phospholipid vesicles. Fluorescence studies using carbazole-labeled and brominated phospholipids

A novel method that uses a carbazole-labeled fluorescent phosphatidylcholine, which partitions preferentially into liquid-crystalline lipid domains, to monitor the kinetics and the extents of thermotropic and ionotropic lateral phase separations in vesicles combining brominated and nonbrominated phosphatidylcholines (PCs), phosphatidic acids (PAs), and phosphatidylserines (PSs) is described. The calcium-induced segregation of several nonbrominated PA species in liquid-crystalline brominated PC bilayers behaves as a well-defined lateral phase separation; the residual solubility of the PA component in the PC-rich phase in the presence of calcium can vary severalfold depending on the PA acyl chain composition. PC/PS mixtures show a pronounced tendency to form metastable solutions in the presence of calcium, particularly when they contain less than equimolar proportions of PS. This metastability is not readily relaxed by repeated freeze-thawing of vesicles in the presence of calcium, by avidin-mediated contacts between PC/PS vesicles containing biotinylated lipids, or by calcium-induced lateral segregation of PA in the same vesicles. Different PS species exhibit different apparent residual solubilities in liquid-crystalline PC bilayers, ranging from less than 10 mol % for dimyristoyl-PS to ca. 45 mol% for dioleoyl-PS, after prolonged incubations of PC/PS multilamellar vesicles with excess calcium. Results are presented, obtained by using the above lipid-segregation assay and parallel assays of intervesicle lipid mixing, that raise questions concerning the relevance of the equilibrium behavior of calcium-treated PS/PC mixtures to the relatively rapid interactions (fusion and lipid mixing) of PC/PS vesicles that follow initial exposure to calcium.     

Dilatometric study of binary mixtures of phosphatidylcholines.

Volumes of lipid dispersions as a function of temperature have been measured for two different kinds of binary mixtures of lecithins, (1) DMPC and DSPC and (2) DMPC and DC20PC. Emphasis was placed on DMPC-rich compositions so as to resolve ambiguities regarding solid-phase immiscibility in DMPC-DSPC mixtures. Special attention has been paid to problems of equilibration in the low-temperature phase and to methods of mixing the lipids. We find that there is no solid-solid immiscibility in DMPC-DSPC mixtures, although this system is close to exhibiting such immiscibility, and that DMPC-DC20PC mixtures exhibit pronounced solid immiscibility.     

Calcium-induced phase separation phenomena in multicomponent unsaturated lipid mixtures

The ability of calcium to induce phase separation in multicomponent lipid mixtures containing various unsaturated species of acidic and neutral phospholipids has been investigated by 31P NMR, 3H NMR, and small-angle X-ray diffraction techniques. It is shown that, in unsaturated (dioleoyl-) phosphatidylglycerol (PG)/phosphatidylethanolamine (PE) (1:1) and phosphatidic acid (PA)/phosphatidylcholine (PC) (1:1) mixtures, calcium is unable to induce lateral phase separation of the acidic and neutral lipids and that all the lipids adopt a hexagonal (HII) phase in the presence of calcium. In multicomponent mixtures containing one or more acidic species the presence of cholesterol either facilitates calcium-induced lamellar to hexagonal (HII) transitions for all the lipid components or, in systems already in a hexagonal (HII) phase, mitigates against calcium-induced lateral phase separations. Further, cholesterol is shown to exhibit no preferential interaction on the NMR time scale with either PC, PE, or phosphatidylserine (PS) when the lipids are in the liquid-crystal state. The ability of cholesterol to directly induce HII phase formation in PC/PE mixtures is also shown to be common to various other sterols including ergosterol, stigmasterol, coprostanol, epicoprostanol, and androstanol.     

Phospholipid dependence of rat brain microsomal glucose-6-phosphate phosphohydrolase

Partial lipid removal of rat brain microsomes by acetone-butanol extraction resulted in 32% loss of activity of glucose-6-phosphate phosphohydrolase (G-6-Pase) and an increase in Km and energy of activation (Ea) of the enzyme while the Vmax was lowered. The activity was restored by supplementation of microsomal total phospholipid (PL) and phosphatidylcholine (PC) in sonicated dispersions but not with neutral lipids, phosphatidyl ethanolamine, sphingomyelin, phosphatidylglycerol and cholesterol. In both intact and delipidated membranes, the activity was decreased by sodium deoxycholate and enhanced by dimethylsulfoxide. Egg yolk PC and asolectin influenced the activity to the extent of that produced by microsomal PC. PC increased the Km of the enzymatic reaction in intact microsomes but decreased the same in disrupted membrane while the Vmax was not affected in both the membranes. Addition of PC into the assay system lowered Ea of the reaction in both the membrane systems. However, there was no break observed in the Arrhenius plot. Ability of liver nonspecific lipid transfer proteins to introduce alien PL into brain microsomes was used to study lipid dependence of G-6-Pase and investigation of membrane-enzyme interrelationship. Protein catalyzed transfer of egg PC from a donor PC-cholesterol unilamellar liposomes resulted in substantial increase in microsomal membrane PC and total PL and a net reduction in the enzyme activity was observed in intact and delipidated membranes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relevance of lipid polar headgroups on boron-mediated changes in membrane physical properties

Using liposomes composed of either brain phosphatidylcholine (PC), or binary mixtures of PC and phosphatidylserine (PS), galactolipids (GL), phosphatidylinositol (PI), cardiolipin (CL), phosphatidic acid (PA), or phosphatidylethanolamine (PE), we investigated the effects of graded amounts of boric acid (B, 0.5-1000 microM) on the following membrane physical properties: (a) surface potential, (b) lipid rearrangement through lateral phase separation, (c) fluidity, and (d) hydration. Incubation of the different populations of vesicles with B was associated with a small, but statistically significant, increase in membrane surface potential in PC, PC:PS, PC:GL, PC:PI, PC:PA, and PC:PE liposomes. B-induced lipid lateral rearrangement through lateral phase separation in PC, PC:PA, and PC:PE liposomes; but had no effects on PC:PS, PC:GL, and PC:PI liposomes. In PC liposomes B affected membrane fluidity at the water-lipid interface without affecting the hydrophobic core of the bilayer. In all the other binary liposomes studied, B increased membrane fluidity in both, the hydrophobic portion of the membrane and in the anionic domains. The above was associated with a decrease in the fluidity of the cationic domains. B (10-1000 microM) decreased membrane hydration regardless the composition of the liposomes. The obtained results demonstrate the ability of B to interact with membranes, and induce changes in membrane physical properties. Importantly, the extent of B-membrane interactions and the consequent effects were dependent on the nature of the lipid molecule; as such, B had greater affinity with lipids containing polyhydroxylated moieties such as GL and PI. These differential interactions may result in different B-induced modulations of membrane-associated processes in cells.     

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)     

Interaction of D-beta-hydroxybutyrate apodehydrogenase with phospholipids.

The interaction of a soluble homogeneous preparation of D-beta-hydroxybutyrate apodehydrogenase with phospholipid was studied in terms of restoration of enzymic activity and complex formation. The purified apoenzyme, which is devoid of lipid, is inactive. It is reactivated specifically by the addition of lecithin or mixtures of phospholipids containing lecithin. Mitochondrial phospholipid, i.e. the mixture of phospholipids in mitochondria, reactivates with the highest specific activity (approximately 100 micromol of DPN reduced/min/mg at 37 degrees and with the greatest efficiency (2.5 to 4 mol of lecithin/mol of enzyme subunit). Each of the lecithins of varying chain length and unsaturation reactivated the enzyme, albeit to differing extents and efficiencies. In general, lecithins containing unsaturated fatty acid moieties reactivated better than those containing the comparable saturated lipid. Optimal reactivation can be obtained for the various lecithins when they are microdispersed together with phosphatidylethanolamine. When the lecithins are added microdispersed together with both phosphatidylethanolamine and cardiolipin, maximal efficiency is obtained. Also, PC6:0 and 8:0 reactivate as soluble molecules, so that a phospholipid bilayer is not necessary to reactivate the enzyme. Complex formation was studied using gel exclusion chromatography. It can be shown that each of the phospholipids which reactivate combines with the apoenzyme. Mitochondrial phospholipid, which reactivates the best, binds most effectively; PC8:0, which reactivates with poor efficiency, can be shown to bind with low affinity, and negligible binding occurs at concentrations which do not reactivate the enzyme. Since the apoenzyme is apparently homogeneous and devoid of phospholipid or detergents, it would appear that reactivation does not involve reversal of inhibition such as by removal of a regulatory subunit or detergent from the catalytic subunit. Rather, we conclude that phospholipid is a necessary and integral portion of this enzyme whose active form is a phospholipid-protein complex. The apoenzyme also forms a complex with phosphatidylethanolamine and/or cardiolipin, which do not reactivate enzymic activity. Salt dissociates such complexes in contrast with the lecithin-apoenzyme complex. Binding of phospholipid is a necessary but not sufficient requisite for enzymic activity. The same energies of activation are obtained from Arrhenius plots for the membrane-bound enzyme and for the purified soluble enzyme reactivated with mitochondrial phospholipid or different lecithins. This observation is compatible with the view that the purified enzyme has not been adversely modified in the isolation. Furthermore, essentially the same energies of activation were obtained for saturated lecithins below their transition temperatures and for unsaturated lecithins above their transition temperatures. Hence, there is no indication that a lipid phase transition occurs to influence the activity of this enzyme.     

Sphingosine inhibits the activity of rat liver CTP:phosphocholine cytidylyltransferase

We report CTP:phosphocholine cytidylyltransferase (CT) as another target enzyme of sphingosine actions in addition to the well-characterized protein kinase C. Effects of sphingosine and lysophingolipids were studied on the activity of purified cytidylyltransferase prepared by the method of Weinhold et al. (Weinhold, P. A., Rounsifer, M.E., and Feldman, D.A. (1986) J. Biol. Chem. 261, 5104-5110). The sphingolipids were tested as components of egg phosphatidylcholine (PC) vesicles, 25 mol% sphingosine inhibited the CT activity by about 50%. The inhibition of CT by sphingosine and lysosphingolipids was reversible. Sphingosine was found to be a reversible inhibitor of CT with respect to the activating lipids such as phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, and fatty acid:phosphatidylcholine vesicles. Egg PC vesicles containing sphingosine, psychosine (galactosylsphingosine), glucopsychosine (glucosylsphingosine), and lysosphingomyelin (sphingosylphosphorylcholine) suppressed the activation by PC/oleic acid vesicles, whereas the parent sphingolipids did not. Egg PC vesicles containing oleylamine and hexadecyltrimethylamine inhibited CT activity, whereas egg PC-octylamine vesicles did not alter the enzyme activity. This indicates the importance of an amino group and long alkyl chain. LysoPC, a known detergent, did not inhibit the enzyme activity under the same assay conditions in which sphingosine inhibited. These results are the first report of a lipid inhibitor of purified CT.     

A rapid separation technique for overcoming suppression of triacylglycerols by phosphatidylcholine using MALDI-TOF MS

Phospholipids and triacylglycerols (TAGs) are important classes of lipids in biological systems. Rapid methods have been developed for their characterization in crude samples, including MALDI time-of-flight MS. For mixtures, MALDI often selectively shows only some components. For example, phosphatidylcholine (PC) suppresses detection of other lipids. Most rapid MS methods detect either TAGs or phospholipids but not both. Herein, we demonstrate a simple approach to rapidly screen mixtures containing multiple lipid classes. To validate this approach, reference lipids [PC, tripalmitin (PPP), and phosphatidyl-ethanolamine (PE)] and real samples (beef, egg yolk) were used. In a binary mixture with a strong suppressor (PC), PPP was greatly suppressed. After a simple separation, suppression was virtually eliminated. A mixture of nominally nonsuppressing lipids (PE and PPP) was not adversely affected by separation. Ground beef and egg yolk were used to demonstrate detection of known lipid compositions where other methods have missed one or more lipids or lipid classes. Separation was performed using solid phase extraction with a PrepSep florisil column. A 10 min separation allows rapid screening for lipids and changes in lipids. It is sufficient to clearly detect all lipids and overcome suppression effects in complex lipid mixtures.     

Effects of dipalmitoylglycerol and fatty acids on membrane structure and protein kinase C activity.

The individual and combined effects of the saturated diacylglycerol (DAG) dipalmitin (DP) and saturated or polyunsaturated unesterified fatty acids (PUFAs) on both the structure of phosphatidylcholine/phosphatidylserine (PC/PS; 4:1 mol/mol) bilayers and on protein kinase C (PKC) activity were studied using 2H nuclear magnetic resonance (NMR) and enzyme activity assays. In the absence of DP, PUFAs only slightly activated PKC whereas palmitic acid had no effect. In the absence of fatty acids, DP induced lateral phase separation of the bilayer into liquid-crystalline and gel phases. Under these conditions virtually all DP was sequestered into the gel phase and no activation of PKC was observed. The addition of polyunsaturated arachidonic or docosahexaenoic acids to the DP-containing bilayers significantly increased the relative amounts of DP and other lipid components in the liquid-crystalline phase, correlating with a dramatic increase in PKC activity. Furthermore, the effect was greater with PS, resulting in an enrichment of PS in the liquid-crystalline domains. In the presence of DP, palmitic acid did not decrease the amount of gel phase lipid and had no effect on PKC activity. The results explain the observed lack of PKC-activating capacity of long-chain saturated DAGs as due to the sequestration of DAG into gel domains wherein it is complexed with phospholipids and thus not available for the required interaction with the enzyme.     

Monte Carlo simulation of lipid mixtures: finding phase separation.

The nonideal mixing of phosphatidylserine (PS) and phosphatidylcholine (PC) binary lipid mixtures was studied by computer simulation based on a model wherein the excess energy of mixing is divided between an electrostatic term and one adjustable term delta Em that includes all other nonideal interactions. The lateral distribution of the lipids and the energy of the mixtures were obtained by using Kawasaki relaxation in a canonical ensemble. The Gibbs free energies were calculated by Kirkwood's coupling parameter method. The simulation results are strongly dependent on simulation size for sizes smaller than about 1000 lipids. Nonideal interaction between lipids can result in large scale separation of lipid phases of different composition at reasonable delta Em values as well as clustering of like lipids. In plots of total Gibbs free energy of mixing versus PS mole fraction in PS/PC, the boundaries of the two phase region could be accurately determined. The electrostatic interaction influences cluster size and shape, and also the composition of phases in the two-phase region.     

The membrane lipid environment modulates drug interactions with the P-glycoprotein multidrug transporter.

The P-glycoprotein multidrug transporter functions as an ATP-driven efflux pump for a large number of structurally unrelated hydrophobic compounds. Substrates are believed to gain access to the transporter after partitioning into the membrane, rather than from the extracellular aqueous phase. The binding of drug substrates to P-glycoprotein may thus be modulated by the properties of the lipid bilayer. The interactions with P-glycoprotein of two drugs (vinblastine and daunorubicin) and a chemosensitizer (verapamil) were characterized by quenching of purified fluorescently labeled protein in the presence of various phospholipids. Biphasic quench curves were observed for vinblastine and verapamil, suggesting that more than one molecule of these compounds may bind to the transporter simultaneously. All three drugs bound to P-glycoprotein with substantially higher affinity in egg phosphatidylcholine (PC), compared to brain phosphatidylserine (PS) and egg phosphatidylethanolamine (PE). The nature of the lipid acyl chains also modulated binding, with affinity decreasing in the order egg PC > dimyristoyl-PC (DMPC) > dipalmitoyl-PC (DPPC). Following reconstitution of the transporter into DMPC, all three compounds bound to P-glycoprotein with 2-4-fold higher affinity in gel phase lipid relative to liquid-crystalline phase lipid. The P-glycoprotein ATPase stimulation/inhibition profiles for the drugs were also altered in different lipids, in a manner consistent with the observed changes in binding affinity. The ability of the drugs to partition into bilayers of phosphatidylcholines was determined. All of the drugs partitioned much better into egg PC relative to DMPC and DPPC. The binding affinity increased (i.e., the value of Kd decreased) as the drug-lipid partition coefficient increased, supporting the proposal that the effective concentration of the drug substrate in the membrane is important for interaction with the transporter. These results provide support for the vacuum cleaner model of P-glycoprotein action.     

The modulation of bovine milk d-galactosyltransferase by various phosphatidylethanolamines

To investigate the possible role of nonbilayer phases in the modulation of glycosyltransferase activity, bovine milk d-galactosyltransferase has been studied in phosphatidylethanolamine (PE) membranes, including soybean PE, egg PE, PE prepared by transphosphatidylation of egg PC, having brain PE, plasmalogen PE, and DPPE. The gel-to-liquid crystalline transition (C_C) and the lamellar-to-hexagonal transitions (T_H) are known for most of the PE compounds. The lower the T_C (or T_H) value, the greater the stimulation of galactosyltransferase activity in both the lactose- and N-acetyllactosamine-synthetase reactions. No correlation was found between either T_C or T_H value and the break in the Arrhenius plots for the N-acetyllactosamine synthetase. In membranes consisting of mixtures of PE with PC, the dominant effect was that of PC. The stimulation of activity in the mixed-lipid systems was never greater than that produced by PC alone, therefore the enzyme showed a definite preference for PC in the mixtures.     

Interaction of rat brain cytidylate cyclase with phospholipids

The interaction of rat brain cytidylate cyclase with some phospholipids such as L-alpha-phosphatidylcholine (PC), L-alpha-phosphatidylserine (PS), L-alpha-phosphatidylethanolamine (PE) and L-alpha-phosphatidic acid (PA) was studied. Cytidylate cyclase activity of Triton X-100 - solubilized fraction was inhibited by PS, PE and PA, but not with PC. The addition of PC to the incubation mixture containing PS, PE or PA dose - dependently reversed the inhibition of enzyme activity by these phospholipids. Phospholipids showed similar effect on the intact membrane - bound enzyme. PC could reactivate the enzyme which was inactivated by deoxycholate treatment, suggesting that PC may be an important factor to reconstitute an active conformation of the enzyme. These findings indicate that cytidylate cyclase could be regulated by phospholipids constituting its microenvironment of the membrane.     

Phosphatidic acid and phosphatidylserine have distinct structural and functional interactions with the nicotinic acetylcholine receptor

Bilayers containing phosphatidylcholine (PC) and the anionic lipid phosphatidic acid (PA) are particularly effective at stabilizing the nicotinic acetylcholine receptor (nAChR) in a functional conformation that undergoes agonist-induced conformational change. The physical properties of PC membranes containing PA are also substantially altered upon incorporation of the nAChR. To test whether or not the negative charge of PA is responsible for this "bi-directional coupling," the nAChR was reconstituted into membranes composed of PC with varying levels of the net negatively charged lipid phosphatidylserine (PS). In contrast to PA, increasing levels of PS in PC membranes do not stabilize an increasing proportion of nAChRs in a functional resting conformation, nor do they slow nAChR peptide hydrogen exchange kinetics. Incorporation of the nAChR had little effect on the physical properties of the PC/PS membranes, as monitored by the gel-to-liquid crystal phase transition temperatures of the bilayers. These results show that a net negative charge alone is not sufficient to account for the unique interactions that occur between the nAChR and PC/PA membranes. Incorporation of the receptor into PC/PS membranes, however, did lead to an altered head group conformation of PS possibly by recruiting divalent cations to the membrane surface. The results show that the nAChR has complex and unique interactions with both PA and PS. The interactions between the nAChR and PS may be bridged by divalent cations, such as calcium.     

Presence of anionic phospholipids rules the membrane localization of phenothiazine type multidrug resistance modulator

Substances able to modulate multidrug resistance (MDR), including antipsychotic phenothiazine derivatives, are mainly cationic amphiphiles. The molecular mechanism of their action can involve interactions with transporter proteins as well as with membrane lipids. The interactions between anionic phospholipids and MDR modulators can be crucial for their action. In present work we study interactions of 2-trifluoromethyl-10-(4-[methanesulfonylamid]buthyl)-phenothiazine (FPhMS) with neutral (PC) and anionic lipids (PG and PS). Using microcalorimetry, steady-state and time-resolved fluorescence spectroscopy we show that FPhMS interacts with all lipids studied and drug location in membrane depends on lipid type. The electrostatic attraction between drug and lipid headgroups presumably keeps phenothiazine derivative molecules closer to surface of negatively charged membranes with respect to neutral ones. FPhMS effects on bilayer properties are not proportional to phosphatidylserine content in lipid mixtures. Behavior of equimolar PC:PS mixtures is similar to pure PS bilayers, while 2:1 or 1:2 (mole:mole) PC:PS mixtures resemble pure PC ones.     

Study of the effect of poly(L-lysine) on phosphatidic acid and phosphatidylcholine/phosphatidic acid bilayers by raman spectroscopy

The effect of polylysine (PLL) on dimyristoylphosphatidic acid (DMPA), on dimyristoyl-phosphatidylcholine (DMPC), and on mixtures of these lipids was investigated by Raman spectroscopy. These results show that long polylysine (Mr approximately 200,000) increases the stability of the acyl chain matrix of DMPA to form a more closely packed structure with a stoichiometry of one lysine residue per PA molecule. On the other hand, short PLL (Mr 4000) destabilizes the PA bilayer, and the complex formed undergoes a gel to liquid-crystalline transition at a lower temperature than of the pure lipid. For both cases, we have observed that bound polylysine adopts a beta-sheet conformation as opposed to the alpha-helical structure previously found for dipalmitoylphosphatidylglycerol/long PLL complexes [Carrier, D., & Pézolet, M. (1984) Biophys. J. 46, 497-506]. The difference in the thermal behavior of complexes of DMPA with long and short polylysines is believed to be associated with the fact that in the complex the long polypeptide adopts the beta-sheet conformation over the whole range of temperatures investigated while the short one undergoes a change of conformation from beta-sheet of random coil upon heating. Therefore, the conformation of the lipid-bound polypeptides depends on the nature of the polar head group of the lipid, not only on its net charge, and it affects considerably the thermotropism of the lipid. On the other hand, both long and short polylysines show no affinity for phosphatidylcholine since the temperature profiles of DMPC and of DMPC/PLL complexes exhibit exactly the same behavior.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of lipids on the transport activity of the reconstituted glucose transport system from rat adipocyte

The glucose transport system, isolated from rat adipocyte membrane fractions, was reconstituted into phospholipid vesicles. Vesicles composed of crude egg yolk phospholipids, containing primarily phosphatidylcholine (PC) and phosphatidylethanolamine (PE), demonstrated specific d-glucose uptake. Purified vesicles made of PC and PE also supported such activity but PC or PE by themselves did not. The modulation of this uptake activity has been studied by systematically altering the lipid composition of the reconstituted system with respect to: (1) polar headgroups; (2) acyl chains, and (3) charge. Addition of small amounts (20 mol%) of PS, phosphatidylinositol (PI), cholesterol, or sphingomyelin significantly reduced glucose transport activity. A similar effect was seen with the charged lipid, phosphatidic acid. In the case of PS, this effect was independent of the acyl chain composition. Polar headgroup modification of PE, however, did not appreciably affect transport activity. Free fatty acids, on the other hand, increased or decreased activity based on the degree of saturation and charge. These results indicate that glucose transport activity is sensitive to specific alterations in both the polar headgroup and acyl chain composition of the surrounding membrane lipids.