Effect of lipid acyl chain length on activity of sodium-dependent leucine transport system in Pseudomonas aeruginosa

The sodium-dependent leucine transport system of Pseudomonas aeruginosa was reconstituted into liposomes of binary lipid mixtures of dilauroylphosphatidylethanolamine (di(12:0)PE)/phosphatidylcholine (PC) with cis-monounsaturated fatty acid chains (di(n:1)PC) (n = 14-22) or dioleoylphosphatidylethanolamine (di(18:1)PE)/di(n:1)PC (n = 14-22). Leucine carrier proteins can be activated with phosphatidylethanolamine, whereas activation does not occur in PC-reconstituted vesicles (Uratani, Y., and Aiyama, A. (1986) J. Biol. Chem. 261, 5450-5454). Na+-dependent counterflow was measured at 30 degrees C as reconstituted transport activity. Proteoliposomes containing di(12:0)PE exhibited high counterflow activity at the PC acyl carbon number (n) of 18 and 20 but no or low activity at n = 14, 16, and 22. On the other hand, proteoliposomes containing di(18:1)PE exhibited higher transport activity than those vesicles with di(12:0)PE and corresponding di(n:1)PC. A lipid mixture of di(18:1)PE and di(16:1)PC supported maximal activity. These results show that the leucine transport system of P. aeruginosa is dependent on the lipid acyl chain length and suggest that there exists optimal bilayer thickness for maximal carrier activity.     

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.     

Acidic phospholipids are required during solubilization of amino acid transport systems of Lactococcus lactis.

The branched-chain amino acid transport system of Lactococcus lactis was solubilized with n-octyl beta-D-gluco-pyranoside and reconstituted into proteoliposomes. Transport activity was recovered only when solubilization was performed in the presence of acidic phospholipids. Omission of acidic phospholipids during solubilization resulted in an inactive transport protein and the activity could not be restored in the reconstitution step. Similar results have been obtained for the arginine/ornithine exchange protein from Pseudomonas aeruginosa and L. lactis. Functional reconstitution of the transport protein requires the presence of aminophospholipids or glycolipids in the liposomes (Driessen, A.J.M., Zheng, T., In't Veld, G., Op den Kamp, J.A.F. and Konings, W.N. (1988) Biochemistry 27, 865-872). We propose that during the detergent solubilization the acidic phospholipids protect the transport systems against denaturation by preventing delipidation.     

Phospholipid effects on SGLT1-mediated glucose transport in rabbit ileum brush border membrane vesicles

In small intestine, sodium-glucose cotransporter SGLT1 provides the main mechanism for sugar uptake. We investigated the effect of membrane phospholipids (PL) on this transport in rabbit ileal brush border membrane vesicles (BBMV). For this, PL of different charge, length, and saturation were incorporated into BBMV. Transport was measured related to (i) membrane surface charge (membrane-bound MC540 fluorescence), (ii) membrane thickness (PL incorporation of different acyl chain length), and (iii) membrane fluidity (r_12AS, fluorescence anisotropy of 12-AS).Compared to phosphatidylcholine (PC) carrying a neutral head group, inhibition of SGLT1 increased considerably with the acidic phosphatidic acid (PA) and phosphatidylinositol (PI) that increase membrane negative surface charge. The order of PL potency was PI>PA > PE = PS > PC. Inhibition by acidic PA-oleate was 5-times more effective than with neutral PE (phosphatidylethanolamine)-oleate. Lineweaver-Burk plot indicated uncompetitive inhibition of SGLT1 by PA.When membrane thickness was increased by neutral PC of varying acyl chain length, transport was increasingly inhibited by 16:1 PC to 22:1 PC. Even more pronounced inhibition was observed with mono-unsaturated instead of saturated acyl chains which increased membrane fluidity (indicated by decreased r_12AS).In conclusion, sodium-dependent glucose transport of rabbit ileal BBMV is modulated by (i) altered membrane surface charge, (ii) length of acyl chains via membrane thickness, and (iii) saturation of PL acyl chains altering membrane fluidity. Transport was attenuated by charged PL with longer and unsaturated acyl residues. Alterations of PL may provide a principle for attenuating dietary glucose uptake.     

Reconstitution of the leucine transport system of Lactococcus lactis into liposomes composed of membrane-spanning lipids from Sulfolobus acidocaldarius.

The effect of bipolar tetraether lipids, extracted from the thermophilic archaebacterium Sulfolobus acidocaldarius, on the branched-chain amino acid transport system of the mesophilic bacterium Lactococcus lactis was investigated. Liposomes were prepared from mixtures of monolayer lipids and the bilayer lipid phosphatidylcholine (PC), analyzed on their miscibility, and fused with membrane vesicles from L. lactis. Freeze-fracture electron microscopy demonstrates that the bipolar lipids in the hybrid membranes adopted a monomolecular organization at high S. acidocaldarius lipid content. Leucine transport activity (i.e., delta mu H(+)-driven and counterflow uptake) increased with the content of S. acidocaldarius lipids and was optimal at a one-to-one (w/w) ratio of PC to S. acidocaldarius lipids. Membrane fluidity decreased with increasing S. acidocaldarius lipid content. These data suggest that transport proteins can be functionally reconstituted into membranes composed of membrane-spanning lipids provided that membrane viscosity is restricted.     

Lipid requirement of the branched-chain amino acid transport system of Streptococcus cremoris

The role of the membrane lipid composition on the transport protein of branched-chain amino acids of the homofermentative lactic acid bacterium Streptococcus cremoris has been investigated. The major membrane lipid species identified in S. cremoris were acidic phospholipids (phosphatidylglycerol and cardiolipin), glycolipids, and glycerophosphoglycolipids. Phosphatidylethanolamine (PE) was completely absent. Protonmotive force-driven and counterflow transport of leucine was assayed in fused membranes of S. cremoris membrane vesicles and liposomes composed of different lipids obtained by the freeze/thaw-sonication technique. High transport activities were observed with natural S. cremoris and Escherichia coli lipids, as well as with mixtures of phosphatidylcholine (PC) with PE or phosphatidylserine. High transport activities were also observed with mixtures of PC with monogalactosyl diglyceride, digalactosyl diglyceride, or a neutral glycolipid fraction isolated from S. cremoris. PC or mixtures of PC with phosphatidylglycerol, phosphatidic acid, or cardiolipin showed low activities. In mixtures of PC and methylated derivatives of PE, both counterflow and protonmotive force-driven transport activities decreased with increasing degree of methylation of PE. The decreased transport activity in membranes containing PC could be restored by refusion with PE-containing liposomes. These results demonstrate that both aminophospholipids and glycolipids can be activators of the leucine transport system from S. cremoris. It is proposed that aminophospholipids in Gram-negative bacteria and glycolipids in Gram-positive bacteria have similar functions with respect to solute transport.     

酿酒酵母酚类抑制物耐受性脂质组学研究

通过脂质组学分析方法从细胞膜磷脂分布方面探究适应进化酿酒酵母酚酸耐受性机制。主要利用高效液相色谱-质谱(LC-MS)对酚酸胁迫下适应进化菌株和原始菌株脂质成分检测并进行统计学比较分析。检测出565种脂质代谢物,包含细胞膜磷脂185种。相比初始菌株,适应进化菌株细胞膜中磷脂酰胆碱(PC)、磷脂酰乙醇胺(PE)和磷脂酰肌醇(PI)类磷脂分子相对含量增加,含有长链(C32-C36)和双不饱和脂酰链的磷脂分子含量增加。统计学分析表明显著性差异磷脂分子主要为含有长链不饱和脂酰链的PC和PE类磷脂分子。推测适应进化菌株通过膜磷脂重塑提高细胞膜完整性,对酚类抑制物起到选择性屏障作用,从而保持细胞活性。     

Depletion of phosphatidylcholine in yeast induces shortening and increased saturation of the lipid acyl chains: evidence for regulation of intrinsic membrane curvature in a eukaryote

To study the consequences of depleting the major membrane phospholipid phosphatidylcholine (PC), exponentially growing cells of a yeast cho2opi3 double deletion mutant were transferred from medium containing choline to choline-free medium. Cell growth did not cease until the PC level had dropped below 2% of total phospholipids after four to five generations. Increasing contents of phosphatidylethanolamine (PE) and phosphatidylinositol made up for the loss of PC. During PC depletion, the remaining PC was subject to acyl chain remodeling with monounsaturated species replacing diunsaturated species, as shown by mass spectrometry. The remodeling of PC did not require turnover by the SPO14-encoded phospholipase D. The changes in the PC species profile were found to reflect an overall shift in the cellular acyl chain composition that exhibited a 40% increase in the ratio of C16 over C18 acyl chains, and a 10% increase in the degree of saturation. The shift was stronger in the phospholipid than in the neutral lipid fraction and strongest in the species profile of PE. The shortening and increased saturation of the PE acyl chains were shown to decrease the nonbilayer propensity of PE. The results point to a regulatory mechanism in yeast that maintains intrinsic membrane curvature in an optimal range.     

Phospholipid turnover and acyl chain remodeling in the yeast ER

The turnover of phospholipids plays an essential role in membrane lipid homeostasis by impacting both lipid head group and acyl chain composition. This review focusses on the degradation and acyl chain remodeling of the major phospholipid classes present in the ER membrane of the reference eukaryote Saccharomyces cerevisiae, i.e. phosphatidylcholine (PC), phosphatidylinositol (PI) and phosphatidylethanolamine (PE). Phospholipid turnover reactions are introduced, and the occurrence and important functions of phospholipid remodeling in higher eukaryotes are briefly summarized. After presenting an inventory of established mechanisms of phospholipid acyl chain exchange, current knowledge of phospholipid degradation and remodeling by phospholipases and acyltransferases localized to the yeast ER is summarized. PC is subject to the PC deacylation-reacylation remodeling pathway (PC-DRP) involving a phospholipase B, the recently identified glycerophosphocholine acyltransferase Gpc1p, and the broad specificity acyltransferase Ale1p. PI is post-synthetically enriched in C18:0 acyl chains by remodeling reactions involving Cst26p. PE may undergo turnover by the phospholipid: diacylglycerol acyltransferase Lro1p as first step in acyl chain remodeling. Clues as to the functions of phospholipid acyl chain remodeling are discussed.     

Microsomal Phospholipid Molecular Species Alterations during Low Temperature Acclimation in Dunaliella

A detailed analysis of the low temperature-induced alterations of Dunaliella salina (UTEX 1644) microsomal membrane lipids was carried out. Microsomal membranes were isolated from cells grown at 30 degrees C, from cells shifted to 12 degrees C for 12 hours, and from cells acclimated to 12 degrees C. Fatty acid analyses of the major lipid classes demonstrated significant changes in the fatty acid composition of phosphatidylcholinemine (PE) and phosphatidylglycerol (PG) but not phosphatidylcholine (PC) during the initial 12 hours at low temperature. These changes did not entail enhanced desaturation of linoleic acid. Subsequent to 12 hours, the proportions of linolenic acid increased in all phospholipids.Molecular species analyses of the phospholipids demonstrated that the most immediate changes following a shift to low temperature were limited to several molecular species of PE and PG. The changes observed in PE included a decrease in C(30) species and concomitant increases in C(34) and C(36) species. Compositional changes associated with PG entailed the emergence of a new molecular species (18:1/18:1) not found at 30 degrees C. The retailoring of molecular species resulted in an increase in the number of species having two unsaturated acyl chains and did not reflect a simple enhancement of desaturase activity as suggested by the fatty acid analysis. We conclude that the initial alterations in response to low temperature stress involve discrete changes in certain molecular species. These and further alterations of molecular species following acclimation to low temperature would appear to augment increases in acyl chain desaturation as a means of modifying membrane properties in response to low temperature stress.     

Phospholipid-Induced Changes of γ-Aminobutyric Acid Transport in Cortex Grey Matter in Culture

The function of membrane phospholipids (PL) in the regulation of gamma-aminobutyric acid (GABA) transport and GABA carrier binding has been investigated in organized cultures of rat cerebral cortex. The cellular lipid composition has been changed by growing the cells in a delipidated nutrient solution or by short-term exposure of the cells to PL emulsions. Introduction of PL into the cellular matrix was monitored by analysis of biologically active fluorescently labeled phosphatidylcholine (PC) or phosphatidylethanolamine (PE). Parinaroyl and dansyl derivatives were used. Conditions of maintenance as well as exogenously given PL affected the transport of GABA. Two transport systems were observed, one first-order system and one cooperative system. Saturated species of PC or PE reduced first-order GABA uptake with increase in chain length of the fatty acid residues. The effects of unsaturated PL were dependent upon the polar head. Unsaturated PC enhanced the capacity of the first-order transport of the amino acid. In comparison to cultures grown in lipid-free medium, introduction of diarachinoyl-PC into the cells increased the density of the first-order active transport sites by a factor of 8 and the affinity constant by a factor of 17. Diarachinoyl-PE reduced both kinetic parameters. GABA uptake via the cooperative system was enhanced by the unsaturated PE, not by PC. The role of endogenous PL and their asymmetric distribution was studied by application of phospholipase A2, C, and D. Stimulation of carrier activity was induced by hydrolysis of PL on the external leaflet. Inhibition occurred upon enzymatic degradation of external and cytoplasmic PL. Lipolysis also affected GABA receptor binding, suggesting that the effects observed represent the activity of both classes of binding sites, the carrier and the receptor. However the latter accounted for a small fraction of the binding. Transport of the amino acid was temperature sensitive. The temperature curve was shifted within two discontinuities, appearing in the Arrhenius plot as a function of membrane lipids. The results suggest a partitioning of the proteins between fluid and ordered lipid domains. Displacement of the protein may govern the rate constants and/or the effective protein concentration.     

膜脂的物理状态对G_s激活AC的影响

用生物膜的拆离与重建方法将从牛脑皮层膜中纯化的激活型ＧＴＰ结合蛋白（Ｇｓ）和腺苷酸环化酶（ＡＣ）在含有不同极性头部或不同脂肪酸侧链的磷脂组成的脂质体上重建形成脂酶体,测定脂酶体中ＡＣ的基础活力及Ｇｓ激活ＡＣ的活力。实验结果表明,磷脂影响ＡＣ的基础活力和Ｇｓ激活ＡＣ活力的顺序依次为：ＰＥ＞ＰＳ＞ＰＣ;含不同脂肪酸侧链的混合磷脂对Ｇｓ的激活活力的影响大于含单一脂肪酸侧链的纯磷脂,如ＰＥＤＰＰＥ,ＰＳＤＰＰＳ,ＰＣＤＰＰＣ。含不同脂肪酸侧链的磷脂影响Ｇｓ的活力的顺序为ＤＬＰＣ＞ＤＭＰＣ＞ＤＰＰＣ。用反映磷脂分子的堆积程度的荧光探剂ＭＣ５４０和脂双层的流动性变化的ＤＰＨ以及专一性标记蛋白质巯基（－ＳＨ）基团的荧光探剂ａｃｒｙｌｏｄａｎ的测定结果表明,不同磷脂影响Ｇｓ的活力的差异主要是由于脂质物理状态的不同所致。     

Effect of membrane fluidity and fatty acid composition on the prothrombin-converting activity of phospholipid vesicles.

Vesicles composed of phospholipids with different fatty acyl side chains have been utilized to examine the importance of the nonpolar membrane region for the prothrombin-converting activity of procoagulant phospholipid vesicles. Membranes composed of phosphatidylserine (PS) and phosphatidylcholine (PC) with unsaturated fatty acyl side chains were more active in prothrombin activation than membranes composed of phospholipids with saturated fatty acyl chains. This phenomenon was observed above the phase transition temperature, i.e., on membranes in the liquid-crystalline state. The prothrombin-converting activity of saturated phospholipids approached the activity of unsaturated phospholipids at high factor Va concentrations, which is indicative for a less favorable equilibrium constant for prothrombinase assembly on membrane surfaces composed of saturated phospholipids. The difference between saturated and unsaturated phospholipids was annulled on membranes with high mole percentages of PS. This may result from a compensating contribution of electrostatic forces to the binding equilibria involved in prothrombinase assembly. Additional effects on the prothrombin-converting activity were observed when membranes containing saturated phospholipids were studied below their phase transition temperature. In agreement with Higgins et al. [(1985) J. Biol. Chem. 260, 3604-3612], we found that the time required for the assembly of prothrombinase from membrane-bound factors Xa and Va is considerably prolonged on solid membranes. However, we also observed an effect of membrane fluidity on the steady-state rate of prothrombin activation. Kinetic experiments at saturating factor Va concentrations showed that the transition from the liquid-crystalline to the gel state caused a more than 9-fold decrease of the kcat of prothrombin activation without affecting the Km for prothrombin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Head group precursors modify phospholipid synthesis in Schistosoma mansoni

The two predominant phospholipids in schistosomula of Schistosoma mansoni are phosphatidylcholine (PC) and phosphatidylethanolamine (PE) which are found in a molar ratio of 0.52 (PE/PC). The incorporation of four fatty acids (arachidonic, myristic, oleic, and palmitic) and glycerol into phospholipids of schistosomula was measured. In two different media (one containing ethanolamine, the other without), all four fatty acids were predominantly incorporated into PC with a PE/PC ratio of approximately 0.1 in a 90-min label. After a 24-h chase, PC remained the predominant labeled phospholipid but the fatty acid-labeled PE/PC ratio increased slightly, the specific activity of labeled neutral lipids decreased, and the specific activity of labeled PE increased. Glycerol was incorporated with a ratio of 0.55 in the presence of ethanolamine but only 0.19 in its absence. Schistosomula also incorporate fatty acids into phosphatidylmonomethylethanolamine (PMME) and phosphatidyldimethylethanolamine (PDME) at rates intermediate to that into PE and PC in the presence of the respective head group precursor; this incorporation was inhibited by choline. Relative to PC, oleic acid is incorporated into PE, PMME, and PDME at rates higher than for palmitic acid. These results suggest that schistosomula possess acyltransferase(s) with head group specificity and that acyl chains are transferred from neutral lipids to phospholipids over time.     

Conformation of fatty acyl chains in alpha- and beta-phosphatidylcholine and phosphatidylethanolamine derivatives in sonicated vesicles

Mono- and dimethylated derivatives constitute important intermediates in the conversion of phosphatidylethanolamine (PE) to phosphatidylcholine (PC) in eucaryote membranes. 1H-NMR techniques were utilized to examine the conformation of the region of the fatty acyl chains that is close to the polar group in the series of alpha-phospholipids: PE, N-methyl-PE, N,N-dimethyl-PE, and PC. The same series of polar groups, but on phospholipid containing sn-1 and/or sn-3 fatty acyl chains (beta-phospholipids) were also examined. All of the phospholipids were in the form of small sonicated vesicles which are widely utilized as membrane models. The alpha-methylene group of the sn-1 and sn-2 fatty acyl chains of the alpha-phospholipids give rise to separate signals due to the non-equivalency of these chains with respect to the glycerol phosphate backbone on all alpha-phospholipids tested. Additionally, differences in the environment of the PC molecules as well as N-methyl-PE, and N,N-dimethyl-PE, but not PE itself on the inside and outside of the vesicles are reflected in the chemical shift of the alpha-methylene protons. On the other hand, all of the beta-phospholipids (including beta-PE) were found to reflect the inside/outside packing differences in their alpha-methylene groups. The bilayer packing does not induce any nonequivalence in the chemically equivalent acyl chains. In mixed micelles with detergents, beta-phospholipids showed one alpha-CH2 signal for all phospholipids. These results are consistent with a common conformational arrangement for the fatty acyl chains in all alpha-phospholipids that have been investigated no matter what aggregated form. The conformational arrangement in the beta-phospholipids is different, but again is similar for all of the compounds tested in various aggregated forms.     

Fluorescence assay of the specificity of human plasma and bovine liver phospholipid transfer proteins

The specificities of a human plasma and bovine liver phospholipid transfer protein were studied using a fluorescence assay based on the transfer of pyrenyl phospholipids. This method was used previously to determine the mechanism of spontaneous transfer of phospholipids between model lipoproteins (Massey, J.B., Gotto, A.M., Jr. and Pownall, H.J. (1982) Biochemistry 21, 3630-3636). The pyrenyl phospholipids varied in the headgroup moiety; pyrenyl phosphatidylcholines contained different fatty acyl chains in the sn-1 position. Model high-density lipoproteins (R-HDL) consisting of apolipoprotein A-I and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) were used as donor and acceptor particles. As previously shown, the bovine liver protein mediated the transfer of only phosphatidylcholine. In contrast, the human plasma protein transferred all species studied which included a phosphatidylserine, phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, phosphatidic acid, sphingomyelin, galactosylcerebroside, and a diacylglycerol. The activity of these transfer proteins was only slightly affected by changes in the acyl chain composition of the transferring lipid. Pyrenyl and radioactive ([3H]POPC) phospholipids were transferred with equal rates by the human transfer protein, suggesting that this protein has similar binding characteristics for pyrenyl and natural phospholipids. Spontaneous phospholipid transfer occurs by the aqueous diffusion of monomeric lipid where the rate is highly dependent on fatty acyl chain composition. In this study, no correlation between the rate of spontaneous transfer and protein-mediated transfer was found. The apparent Km values for R-HDL and low-density lipoprotein (LDL), when used as acceptors, were similar when based on the number of acceptor particles. The apparent Vmax for the bovine liver protein was identical for R-HDL and LDL but for the plasma protein Vmax was slightly higher for R-HDL. These results suggest that, like the bovine liver protein, the plasma protein functions as a phospholipid-binding carrier that exchanges phospholipids between membrane surfaces. The assay of lipid transfer proteins by pyrenyl-labeled lipids is faster and easier to perform than other current methods, which require separation of donor and acceptor particles, and is suitable for studies on the function and mechanism of action of lipid transfer proteins.     

Turnover of phospholipid fatty acyl chains in cultured neuroblastoma cells: involvement of deacylation-reacylation and de novo synthesis in plasma membranes

Cultured neuroblastoma cells (NIE-115) rapidly incorporated the essential fatty acid, linoleic acid (18:2 (n = 6), into membrane phospholipids. Fatty acid label appeared rapidly (2-10 min) in plasma membrane phospholipids without evidence of an initial lag. Specific activity (nmol fatty acid/mumol phospholipid) was 1.5-2-fold higher in microsomes than in plasma membrane. In these membrane fractions phosphatidylcholine had at least 2-fold higher specific activity than other phospholipids. With 32P as radioactive precursor, the specific activity of phosphatidylinositol was 2-fold higher compared to other phospholipids in both plasma membrane and microsomes. Thus a differential turnover of fatty acyl and head group moieties of both phospholipids was suggested. This was confirmed in dual-label (3H fatty acid and 32P), pulse-chase studies that showed a relatively rapid loss of fatty acyl chains compared to the head group of phosphatidylcholine; the opposite occurred with phosphatidylinositol. A high loss of fatty acyl chain relative to phosphorus indicated involvement of deacylation-reacylation in fatty acyl chain turnover. The patterns of label loss in pulse-chase experiments at 37 and 10 degrees C indicated some independent synthesis and modification of plasma membrane phospholipids at the plasma membrane. Lysophosphatidylcholine acyltransferase and choline phosphotransferase activities were demonstrated in isolated plasma membrane in vitro. Thus, studies with intact cells and with isolated membrane fractions suggested that neuroblastoma plasma membranes possess enzyme activities capable of altering phospholipid fatty acyl chain composition by deacylation-reacylation and de novo synthesis at the plasma membrane itself.     

Phosphatidylcholine fluidity and structure affect lecithin:cholesterol acyltransferase activity

The purpose of this study was to test the hypothesis that lipid fluidity regulates lecithin:cholesterol acyltransferase (LCAT) activity. Phosphatidylcholine (PC) species were synthesized that varied in fluidity by changing the number, type (cis vs. trans), or position of the double bonds in 18 or 20 carbon sn-2 fatty acyl chains and recombined with [(3)H]cholesterol and apolipoprotein A-I to form recombinant high density lipoprotein (rHDL) substrate particles. The activity of purified human plasma LCAT decreased with PC sn-2 fatty acyl chains containing trans versus cis double bonds and as double bonds were moved towards the methyl terminus of the sn-2 fatty acyl chain. The decrease in LCAT activity was significantly correlated with a decrease in rHDL fluidity (measured by diphenylhexatriene fluorescence polarization) for PC species containing 18 carbon (r(2) = 0.61, n = 18) and 20 carbon (r(2) = 0.93, n = 5) sn-2 fatty acyl chains. rHDL were also made containing 10% of the 18 carbon sn-2 fatty acyl chain PC species and 90% of an inert PC ether matrix (sn-1 18:1, sn-2 16:0 PC ether) to normalize rHDL fluidity. Even though fluidity was similar among the PC ether-containing rHDL, the order of PC reactivity with LCAT was significantly correlated (r(2) = 0.71) with that of 100% PC rHDL containing the same 18 carbon sn-2 fatty acyl chain species, suggesting that PC structure in the active site of LCAT determines reactivity in the absence of measurable differences in bilayer fluidity. We conclude that PC fluidity and structure are major regulators of LCAT activity when fatty acyl chain length is constant.     

Phosphatidylethanolamine and phosphatidylcholine are sources of diacylglycerol in ras-transformed NIH 3T3 fibroblasts

Ras-transformation of cells is accompanied by an increase of the level of diacylglycerol (DAG), which participates in the signal transduction pathways. DAG could be generated from phospholipids either by activation of phospholipase C or by a more complex pathway involving phospholipase D and phosphatidate phosphohydrolase. To clarify which phospholipids produce DAG and which pathways are involved, we examined the DAG generating enzyme activities, using phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylinositol (PI) as substrates. The study showed that the breakdown of PC and more markedly of PE by phospholipases C and D was stimulated in membranes from ras-transformed cells. Phosphatidate phosphohydrolase activity was also elevated in oncogene-expressing cells. The increase in glycerol uptake was most pronounced in cells given PE, followed by PC. The fatty acid analysis revealed apparent similarities between the acyl chains of PE and DAG only in the transformed cells. These findings suggest that PE is a source of DAG in ras-fibroblasts but does not rule out the role of PC in DAG production, due to the activation of the PC-specific phospholipases C and D.