A comparative fluorescence polarization study of cis-parinaroyl-phosphatidylcholine and diphenylhexatriene in membranes containing different amounts of cholesterol

The steady state fluorescence anisotropy (rs) of 1-acyl-2-cis parinaroyl phosphatidylcholine (PnPC) was compared with that of diphenylhexatriene (DPH) in a variety of model- and biological membrane systems. The fluorescence anisotropy of both probes responded similarly to temperature changes and variations in the acyl chain composition in phosphatidylcholine (PC) liposomes. The presence of proteins and cholesterol increased rs for both DPH and PnPC in the biological membranes as compared to the isolated polar membrane lipids. Comparison of DPH and PnPC in dipalmitoyl-PC-liposomes with and without 50 mol% cholesterol, showed at temperatures above the phase transition of pure dipalmitoyl-PC the presence of cholesterol increased the rs-value for DPH strongly, whereas the rs-value for PnPC was much less affected. In the cholesterol-rich erythrocyte membrane as well as in microsomes from Morris hepatoma 7787, which have an increased cholesterol content as compared to normal rat liver microsomes, the rs of DPH was higher than that of PnPC. No large differences between the rs-values of both probes were evident in the normal cholesterol-poor rat liver microsomes. These effects are discussed in terms of structural differences between the probes and variation of cholesterol content. Alterations in the fatty acid composition of PC present in human erythrocyte membranes were introduced with the aid of a PC-specific transfer protein. Fluorescence anisotropy values of both probes hardly changed upon enrichment of the red cell membrane with either dipalmitoyl PC or 1-palmitoyl-2-arachidonyl PC.     

The phosphatidylethanolamine N-methyltransferase pathway is quantitatively not essential for biliary phosphatidylcholine secretion

The phosphatidylethanolamine N-methyltransferase (PEMT) pathway of phosphatidylcholine (PC) biosynthesis is not essential for the highly specific acyl chain composition of biliary PC. We evaluated whether the PEMT pathway is quantitatively important for biliary PC secretion in mice under various experimental conditions. Biliary bile salt and PC secretion were determined in mice in which the gene encoding PEMT was inactivated (Pemt(-/-)) and in wild-type mice under basal conditions, during acute metabolic stress (intravenous infusion of the bile salt tauroursodeoxycholate), and during chronic metabolic stress (feeding a taurocholate-containing diet for 1 week). The activity of CTP:phosphocholine cytidylyltransferase, the rate-limiting enzyme of PC biosynthesis via the CDP-choline pathway, and the abundance of multi-drug-resistant protein 2 (Mdr2; encoded by the Abcb4 gene), the canalicular membrane flippase essential for biliary PC secretion, were determined. Under basal conditions, Pemt(-/-) and wild-type mice exhibited similar biliary secretion rates of bile salt and PC ( approximately 145 and approximately 28 nmol/min/100 g body weight, respectively). During acute or chronic bile salt administration, the biliary PC secretion rates increased similarly in Pemt(-/-) and control mice. Mdr2 mRNA and protein abundance did not differ between Pemt(-/-) and wild-type mice. The cytidylyltransferase activity in hepatic lysates was increased by 20% in Pemt(-/-) mice fed the basal (bile salt-free) diet (P < 0.05). We conclude that the biosynthesis of PC via the PEMT pathway is not quantitatively essential for biliary PC secretion under acute or chronic bile salt administration.     

Phosphatidylcholine and cholesterol interactions in model membranes

Various phosphatidylcholines differing either in the stereochemistry around their chiral center or in the position of a cis double bond along the acyl chains were synthesized in order to study critical contact regions in the phospholipid molecule with adjacent cholesterol in model membranes. Microviscosities calculated from fluorescence depolarization of diphenylhexatriene and chain order from spin label studies were measured to monitor physical membrane properties. The enhancing effect of cholesterol on the microviscosity of membranes containing phosphatidylcholines with comparable acyl chain length was largest when the two acyl chains were saturated and smallest when both were unsaturated. Membranes prepared from phosphatidylcholines having a single cis double bond at different positions along the sn-2 acyl chain showed roughly the same changes of microviscosity or chain order upon incorporation of cholesterol. No discrimination was evident in the interaction between cholesterol and enantiomeric phosphatidylcholines or between the enantiomeric phosphatidylcholine molecules themselves. We conclude that the rigidifying effect of cholesterol in membranes does not depend on specific sites of interaction and that with respect to physical membrane properties phosphatidylcholine behaves as an achiral molecule.     

Involvement of LEM3/ROS3 in the Uptake of Phosphatidylcholine with Short Acyl Chains in Saccharomyces cerevisiae

A Saccharomyces cerevisiae strain deficient in phosphatidylethanolamine methyltransferase that exhibits choline auxotrophy grew in the presence of dioctanoyl phosphatidylcholine (diC8PC). We isolated and characterized mutants defective in growth in the diC8PC-containing medium. Mutations in LEM3/ROS3 impaired growth in that medium, indicating that Lem3p is involved in the utilization of extracellular phosphatidylcholine (PC) with short acyl chains.     

An analysis of the regions of the myelin basic protein that bind to phosphatidylcholine

The interactions of phosphatidylcholine (PC) to regions of the myelin basic protein (MBP) was examined. In solid phase binding assays the nature of the binding of unilamellar vesicles of¹⁴C-labeled phosphatidylcholine to bovine 18.5 kDa MBP, its N- and C-terminal peptide fragments, photooxidized 18.5 kDa MBP and the mouse 14 kDa protein, with an internal deletion of residues 117–157, was studied. The data were analyzed by computer-generated Scatchard plots in which non-specific binding was eliminated. Non-cooperative, low affinity binding of PC vesicles to MBP was observed, and this binding found to be sensitive to pH and ionic changes. At an ionic strength of 0.1 and pH 7.4, the binding of PC to the 14 kDa mouse MBP exhibited a K_d similar to that obtained with both the N-terminal and photooxidized 18.5 kDa bovine MBP. The studies indicated that the sites of PC interaction with MBP are located in the N-terminal region of the protein. The C-terminal region appeared to modulate the strength of the interaction slightly. Under similar conditions, lysozyme did not bind PC liposomes, and histone bound them nonspecifically.     

Phosphatidylcholine’s functions beyond that of a membrane brick

Abstract

Since its discovery in the 19th century, phosphatidylcholine (PC) has been regarded primarily as a structural lipid. However, more recent evidence, much of it in the last five years, strongly suggests that PC has other roles. Here, we explore some of that new evidence and consider the possibility that the ultimate role of phosphatidylcholine may not be predictable.     

A phylogenetic survey of biliary lipids in vertebrates

Biliary lipids (bile salts, phospholipids, cholesterol, plant sterols) were determined in 89 vertebrate species (cartilaginous and bony fish, reptiles, birds, and mammals), and individual phospholipid classes were measured in 35 species. All samples contained conjugated bile salts (C(27) bile alcohol sulfates and/or N-acyl amidates of C(27) and/or C(24) bile acids). Phospholipids were generally absent in the bile of cartilaginous fish and reptiles and were present in low amounts relative to bile salts in bony fish and most birds. In mammals, the phospholipid-bile salt ratio varied widely. The bile from species with low biliary phospholipid-bile salt ratios often contained a high proportion of sphingomyelin, confirmed by HPLC-MS. In species with a high phospholipid-bile salt ratio, the predominant biliary phospholipid was phosphatidylcholine (PC). The phospholipid-bile salt ratio correlated weakly with the calculated weighted hydrophobic index value. Cholesterol was present in the bile of virtually all species, with plant sterols uniformly being present in only trace amounts. The cholesterol-bile salt ratio tended to be higher in mammals than in non-mammals, but bile of all species was unsaturated. Thus, most nonmammalian vertebrates have relatively low levels of biliary phospholipid and cholesterol, suggesting that cholesterol is eliminated predominantly as bile salts. Mammals have a higher phospholipid and cholesterol to bile salt ratio, with the dominant phospholipid being PC.     

Liposomal membranes XI. A suggestion to structural characteristics of acido-thermophilic bacterial membranes

To understand the role of ω-cyclohexyl fatty acid residue of lipids in acido-thermophilic bacterial membranes, three unusual phosphatidylcholines, 1,2-di-11-cyclohexylundecanoyl-l-α-phosphatidylcholine (11_CYPC), 1,2-di-13-cyclohexyltridecanoyl-l-α-phosphatidylcholine (13_CYPC), and 1–13-cyclohexyltridecanoyl-2–11-cyclohexylundecanoyl-l-α-phosphatidylcholine (1–13_CY-2–11_CYPC) were prepared and the steady-state fluorescence anisotropy of 1,6-diphenylhexatriene (DPH) in the hydrophobic domain of these liposomal bilayers was determined. Compared with the case of dipalmitoyl (DPPC) or dimyristoyl phosphatidylcholine (DMPC), introducing the ω-cyclohexyl moiety onto lecithins makes the bilayers fluid below the phase transition temperature, while immobilizes them above the phase transition temperatures. The properties of the unusual phosphatidylcholine liposomes suggested by the steady-state fluorescence anisotropy investigation were in good agreement with those obtained from the thermotropic and permeability investigations. Results obtained are discussed from the view point of the role and function of lipid membranes of acido-thermophilic bacteria which contain unusual fatty acids.     

Synthesis and hemocompatibity evaluation of segmented polyurethane end-capped with both a fluorine tail and phosphatidylcholine polar headgroups

To improve the hemocompatibility of polyurethanes, an amine monomer containing a long fluorine tail and phosphatidylcholine polar headgroups, 2-amino-3-oxo-3-(2-(2,2,3,3,4,4,5,5,6,6,7,7,8,8,8-pentadecafluorooctan amido) ethyl amino) propyl phosphorylcholine (FASPC) was firstly synthesized and characterized. Then four kinds of fluorinated phosphatidylcholine end-capped polyurethanes with different chemical structures were prepared. The surface properties of these prepared polyurethanes were characterized using X-ray photoelectron spectroscopic analysis (XPS) and water contact angle measurements. The results indicated that the phosphatidylcholine (PC) polar headgroups along with the fluorine tail could be easily enriched on the top surfaces, and the PC groups could be highly oriented on the outmost surface when the polymer film was in contact with water for only 30 s at room temperature. The evaluation of hemocompatibity was carried out via fibrinogen adsorption and platelet adhesion. Fibrinogen adsorption (37°C for 90 min) decreased by 98% to 87% compared to that on ordinary polyurethane surfaces, and almost no platelet adhesion and activation was observed at 37°C for 2 h.     

Oxidatively Modified Plasma Phospholipids Containing Reactive Carbonyl Functions Measured by HPLC: Evidence for Phosphatidylcholine-Bound Aldehydes in Plasma of Burn Patients

A HPLC method has been developed to measure phosphatidylcholine (PC) containing reactive carbonyl functions in the sn-acyl residue in order to study processes in which such reactive carbonyls can be formed due to e.g. oxidative fragmentation. The method has been applied to determine PC-bound carbonyls as 2, 4-dinitrophenylhydrazones (DNPH) in plasma of burn patients. Plasma from healthy volunteers served as controls. Additionally, in vitro oxidation experiments (A: plasma, buffer diluted; B: plasma + iron-EDTA complex and C: plasma + iron-EDTA complex + H₂O₂) have been performed to obtain and to identify 2, 4-dinitrophenylhydrazine derivatizable carbonyl functions in plasma PC. Both, the PC-aldehydes and PC-aldehyde DNPH derivatives were cleavable with phospholipase C. Quantification was based on thin-layer chromatography purified soybean phosphatidylcholine, which was identically oxidized and derivatized as the plasma lipids in vitro.     

Theaflavin-3-gallate specifically interacts with phosphatidylcholine,forming a precipitate resistant against the detergent action of bile salt

Black tea is a highly popular beverage, and its pigments, polymerized catechins such as theaflavins (TFs), are attracting attention due to their beneficial health effects. In this study, to test the inhibitory activities of TFs on the intestinal absorption of cholesterol, we investigated their effects on phosphatidylcholine (PC) vesicles in the absence or presence of a bile salt. (?)-Epicatechin gallate, (?)-epigallocatechin gallate, and TFs formed insoluble complexes with PC vesicles. Galloylated TFs such as TF2A, TF2B, and TF3 precipitated far more than other polyphenols. The subsequent addition of taurocholate redispersed the polyphenol-PC complexes, except that a large amount of TF2A remained insoluble. After incubation with taurocholate-PC micelles, TF2A elevated the turbidity of the micelle solution, providing red sediments. The TF2A-specific effect was dependent on the PC concentration. These results suggest that TF2A interacts with PC and aggregates in a specific manner different from catechins and other TFs.     

Spontaneous reconstitution of discoidal HDL from sphingomyelin-containing model membranes by apolipoprotein A-I

Nascent HDL is known to be formed by the interaction of apolipoprotein A-I (apoA-I) with transmembrane ABCA1, but the molecular mechanism by which nascent HDL forms is less well understood. Here, we studied how reconstituted high density lipoprotein (rHDL) forms spontaneously on the interaction of apoA-I with model membranes. The formation of rHDL from pure phosphatidylcholine (PC) large unilamellar vesicles (LUVs) proceeded very slowly at 37.0 degrees C, but sphingomyelin (SM) -rich PC/SM LUVs, which are in a gel/liquid-disordered phase (L(d) phase) at this temperature, were rapidly microsolubilized to form rHDL by apoA-I. The addition of cholesterol decreased the rate at which rHDL formed and induced the selective extraction of lipids by apoA-I, which preferably extracted lipids of L(d) phase rather than lipids of liquid-ordered phase. In addition, apoA-I extracted lipids from the outer and inner leaflets of LUVs simultaneously. These results suggest that the heterogeneous interface of the mixed membranes facilitates the insertion of apoA-I and induces L(d) phase-selective but leaflet-nonselective lipid extraction to form rHDL; they are compatible with recent cell works on apoA-I-dependent HDL generation.     

Ageing-Induced Alterations in Lipid/Phospholipid Profiles of Rat Brain and Liver Mitochondria: Implications for Mitochondrial Energy-Linked Functions

Effects of ageing on the lipid/phospholipid profile of brain and liver mitochondria from rats were examined. In the brain mitochondria the contents of total phospholipid (TPL) and cholesterol (CHL) increased with simultaneous increase in the TPL/CHL (mole:mole) ratio. The proportion and contents of lysophospholipid (Lyso), sphingomyelin (SPM), phosphatidylinositol (PI), phosphatidylserine (PS) and diphosphatidylglycerol (DPG) components increased, with maximal increases seen for PS and PI; phosphatidylcholine (PC) and phosphatidylethanolamine (PE) components registered decrease. In the liver mitochondria contents of TPL and CHL increased. However, the TPL/CHL (mole:mole) ratio was not altered. Lyso, PI and PS increased. However, the magnitude of increase was competitively lower; PE and DPG decreased. SPM and PC did not change as a consequence of ageing. These changes altered the contents of individual phospholipids in the two membrane systems. Respiration with glutamate, pyruvate + malate, succinate and ascorbate + N,N,N’,N’-tetramethyl-p-phenylenediamine was significantly impaired in brain mitochondria from old animals. For liver mitochondria the respiratory activity declined with glutamate and succinate. Correlation studies by regression analysis revealed that the lipid/phospholipid classes regulate respiratory function differently in the mitochondria from the two tissues. The respiration-related parameters in the brain mitochondria were dependent on multiple lipid/phospholipid components, and the process of regulation was complex compared to the liver mitochondrial functions.     

Incorporation of cholesterol into serum high density lipoprotein apoprotein and recombinants

The uptake of cholesterol (CHL) by serum high density lipoprotein (HDL) delipidated apoproteins and phospholipid-apoprotein recombinants has been studied with two methods; by incubation with Celite-dispersed cholesterol or with cholesterol crystals. The apoproteins bind very small amounts of cholesterol with a maximum of about 6 micrograms/mg apoprotein. Recombinants with dimyristoyl phosphatidylcholine (DMPC) or egg phosphatidylcholine (EPC) as phospholipid component gave similar values for cholesterol uptake. The initial rate for uptake from Celite-cholesterol by recombinants was high (0.1 mol cholesterol/mol phospholipid/h) and somewhat higher than that for phospholipid vesicles. The maximal uptake was by gel filtration shown to depend on the size of the complexes with values about 0.95 mol cholesterol per phospholipid for vesicular complexes, 0.75 for discoidal complexes and between 0.5 and 0.2 for small 'protein-rich' complexes. During the incubation of recombinants with cholesterol there was considerable decomposition of discoidal complexes and formation of larger ones. The results show that phospholipid-apoprotein complexes are efficient acceptors for cholesterol but also that about 25% of the phospholipid in the discoidal complexes is excluded from interaction with cholesterol by interaction with apoprotein.     

In vitro synthesis of phosphatidylinositol and phosphatidylcholine by phospholipase D

Phosphatidylinositol (PI) was prepared from egg lecithin by a one-step transphosphatidylation reaction catalysed by phospholipase D in the presence of myo-inositol. Similarly phosphatidylcholine (PC) has been synthesized by the same technique from egg phosphatidylethanolamine using phospholipase D and choline chloride.The yield of PI was ca 25 % and that of PC ca 28 %. The transphosphatidylase function of phospholipase D offers a useful route for the synthesis of different classes of phospholipids.     

反相高效液相色谱法分离肝源性磷脂酰胆碱分子种

建立了反相高效液相色谱法(RP-HPLC)分离肝源性磷脂酰胆碱(PC)分子种的有效方法。考察了流动相中有机溶剂的种类、配比及流速对分离肝源性PC的影响。研究发现,在以甲醇为主的流动相中加入正己烷和醋酸铵有利于肝源性PC分子种的分离;甲醇-乙腈-水梯度洗脱不适于分离肝源性PC分子种。结果表明,采用Kromasil C18色谱柱(4.6 mm×200 mm,5μm),以甲醇-正己烷-0.05 mol/L醋酸铵-甘油(84∶6∶8∶0.6,体积比)为流动相,在流速1.0 mL/min、检测波长206 nm、柱温35℃的条件下,实现了肝源性PC各组分的分离。所建立的方法灵敏,重复性高,为进一步采用液质联用技术研究肝源性PC不同分子种的结构奠定基础。     

Synthesis of phosphatidylcholine and phosphatidylglycerol in rat lung mitochondria

Summary The mitochondrial fraction of adult rat lung contains choline phosphotransferase (EC 2.7.8.2) activity which can not be explained by microsomal contamination estimated on the basis of marker enzyme distribution. Mitochondrial (¹⁴C)glycerol-3-phosphate incorporation into PC (phosphatidylcholine) can be distinguished from the microsomal incorporation by different sensitivity to N-ethylmaleimide inhibition. The data indicate that rat lung mitochondria have the intrinsic capability to synthesize PC. Both synthesis of PC and PG (phosphatidylglycerol) are susceptible to isotonic tryptic attack against the cytoplasmic face of isolated rat lung mitochondria, suggesting the outer membrane location of crucial activities involved in the formation of these phospholipids. Rat liver mitochondria are different from rat lung mitochondria with respect to their capability to synthesize PC, their rate of (¹⁴C)glycerol-3-phosphate incorporation into PG as well as the submitochondrial site of PG formation.Abbreviations PC Phosphatidylcholine - PG Phosphatidylglycerol - PA Phosphatidic Acid - DPG Diphosphatidylglycerol (cardiolipin) - CPT Choline Phosphotransferase (EC 2.7.8.2) - SEM Standard Error of Mean     

The membrane of intact human erythrocytes tolerates only limited changes in the fatty acid composition of its phosphatidylcholine

(1) Using the phosphatidylcholine specific transfer protein from bovine liver, native phosphatidylcholine from intact human erythrocytes was replaced by a variety of different phosphatidylcholine species without altering the original phospholipid and cholesterol content. (2) The replacement of native phosphatidylcholine by the disaturated species, 1,2-dipalmitoyl- and 1,2-distearoylphosphatidylcholine, proceeded at a low rate and extensive replacement could only be achieved by repeatedly adding fresh donor vesicles. The replacement by disaturated molecules was accompanied by a gradual increase in osmotic fragility of the cells, finally resulting in hemolysis when 40% of the native PC had been replaced. Up to this lytic concentration, the replacement did not affect the permeability of the membrane for potassium ions. (3) Essentially, all of the PC in the outer monolayer of the membrane could be replaced by 1-palmitoyl-2-oleoyl- and 1-palmitoyl-2-linoleoylphosphatidylcholine. These replacements did not alter the osmotic fragility of the cells, nor the K⁺ permeability of the membrane. (4) Increasing the total degree of unsaturation of the phosphatidylcholine species modified the properties of the membrane considerably. Replacement by 1,2-dilinoleoylphosphatidylcholine resulted in a progressive increase in osmotic fragility and hemolysis started to occur after 30% of the native PC had been replaced by this species. K⁺ permeability was found to be slightly increased in this case. Cells became leaky for K⁺ upon the introduction of 1-palmitoyl-2-arachidonoylphosphatidylcholine in the membrane. The increased permeability was also reflected by an apparent increase in the resistance of the cells against osmotic shock. (5) The conclusions to be drawn are that (i) 1-palmitoyl-2-oleoyl- and 1-palmitoyl-2-linoleoylphosphatidylcholine are species which fit most optimally into the erythrocyte membrane; (ii) loss of membrane stability results from an increase in the degree of saturation of phosphatidylcholine ($\text{unsaturation index}\text{> 0.5}$) and (iii) the permeability is enhanced by increasing the content of highly unsaturated species ($\text{unsaturation index}\text{> 1.0}$).     

Particle size interconversion of human low density lipoproteins during incubation of plasma with phosphatidylcholine vesicles

Incubation of plasma (37°C, 6hr) in the presence of increasing amounts of phosphatidylcholine (PC) vesicles, above a threshold concentration, results in an increase in particle diameter of LDL relative to that from nonincubated plasma. With further PC addition, the major peak of LDL in the gradient gel electrophoretic pattern is transformed, first, into a bimodal and, subsequently, into a single peak distribution. PC-induced interconversion of LDL requires factors(s) in the d > 1.20 g/ml fraction and, at PC concentrations below approximately 2 mg/ml, is not inhibited by p-chloromercuriphenylsulfonic acid. Plasma incubation with increasing PC levels also leads to characteristic particle size transformations in HDL₃ species, with the transformation products ultimately converging to form a single peak pattern within the HDL_2a size interval. In certain subjects, incubation of plasma, in the absence of added PC, shifts the particle size distribution of LDL towards smaller species; this can be prevented by addition of PC. We propose that incubation-induced shifts of LDL towards larger or smaller species result from changes in phospholipid (PL) content of LDL.     

Uptake and metabolism of lipoprotein-X in mesangial cells

Progressive glomerulosclerosis is a major complication in patients with familial lecithin:cholesterol acyltransferase (LCAT) deficiency. The lack of LCAT activity results in the accumulation of an abnormal lipoprotein, lipoprotein-X (Lp-X), in the plasma of these patients. Lipoprotein-X contains high levels of unesterified cholesterol and phosphatidylcholine. Lp-X may play a role in the accumulation of lipids in the kidney, which in turn may lead to glomerulosclerosis. The objective of this study is to examine the uptake and metabolism of Lp-X by rat mesangial cells. Our results suggest that Lp-X is taken up by mesangial cells and that the lipids in Lp-X are metabolized. Lysosomes containing unesterified cholesterol and phosphatidylcholine, in a molar ratio similar to Lp-X, were synthesized to investigate the roles individual apolipoproteins (apo CI, II, III and E) play in the uptake of Lp-X. Both apo CI and CIII inhibited its uptake while apo CII (1.5 fold) and E (4 fold) stimulated t he uptake of Lp-X. Very low density lipoprotein (VLDL) and low density lipoprotein (LDL) inhibited Lp-X uptake by mesangial cells. However, at higher concentrations of high density lipoprotein (HDL), the uptake of Lp-X was stimulated. Proteoglycans have an important role in regulating the uptake of Lp-X, while cytoskeleton-dependent phagocytosis and the scavenger receptor do not appear to be involved. (Mol Cell Biochem 175: 187–194, 1997)