Interaction of human plasma lecithin:cholesterol acyltransferase and venom phospholipase A2 with apolipoprotein A-I recombinants containing nonhydrolyzable diether phosphatidylcholines

Partially reassembled high density lipoproteins (R-HDL) composed of apolipoprotein A-I and nonhydrolyzable analogues of phosphatidylcholine have been prepared, and their physical properties and reactivities as substrates for lecithin: cholesterol acyltransferase and three phospholipases were tested. The stereo-chemical pairs L-DMPC-ether (1,2-O-ditetradecyl-sn-glycero-3-phosphorylcholine) and D-DMPC-ether (2,3-O-ditetradecyl-sn-glycero-1-phosphoryline) or L-DMPC (1,2-dimyristoyl-sn-glycero-3-phosphoryl-choline) and D-DMPC (2,3-dimyristoyl-sn-glycero-1-phosphorylcholine) have similar thermal properties. R-HDL composed of these four lipids also have similar thermal properties as well as lipid/protein ratios, molecular weights, and protein conformations. Vmax and apparent Km values for lecithin: cholesterol acyltransferase on R-HDL consisting of linear combinations of L-DMPC and D-DMPC, L-DMPC-ether, or D-DMPC-ether plus 6 mol % cholesterol were measured. For the ether lecithins, there was a linear increase in Vmax with percentage of the acyl donor, L-DMPC, in R-HDL; over the same range, there was no change in Km. A comparison with bee venom and Naja melanoleuca phospholipase A2 demonstrated that the venom enzymes have turnover numbers almost 3 orders of magnitude greater than has lecithin:cholesterol acyltransferase; the activity of the phospholipases was profoundly affected by the physical state of the lipid, whereas lecithin: cholesterol acyltransferase activity was not. The differences between these two types of enzymes, which cleave the same bonds of a phosphatidylcholine, are assigned to different catalytic mechanisms. These studies show that R-HDL containing sn-glycero-3-phosphorylcholines and sn-glycero-3-phosphorylcholine ethers have similar structure, properties, and affinities for phospholipolytic enzymes.     

Phospholipid fatty acid modification of rat liver microsomes affects acylcoenzyme A:cholesterol acyltransferase activity

The effect of phospholipid fatty acyl composition on the activity of acylcoenzyme A:cholesterol acyltransferase was investigated in rat liver microsomes. Specific phosphatidylcholine replacements were produced by incubating the microsomes with liposomes and bovine liver phospholipid-exchange protein. Although the fatty acid composition of the microsomes was modified appreciably, there was no change in the microsomal phospholipid or cholesterol content. As compared to microsomes enriched for 2 h with dioleoylphosphatidylcholine, those enriched with dipalmitoylphosphatidylcholine exhibited 30-45% less acyl-CoA:cholesterol acyltransferase activity. Enrichment with 1-palmitoyl-2-linoleoylphosphatidylcholine increased acyl-CoA:cholesterol acyltransferase activity by 20%. By contrast, dilinoleoylphosphatidylcholine abolished microsomal acyl-CoA:cholesterol acyltransferase activity almost completely. Addition of cofactors that stimulated microsomal lipid peroxidation inhibited acyl-CoA:cholesterol acyltransferase activity by only 10%, however, and did not increase the inhibition produced by submaximal amounts of dilinoleoylphosphatidylcholine. Certain of the phosphatidylcholine replacements produced changes in palmitoyl-CoA hydrolase, NADPH-dependent lipid peroxidase, glucose-6-phosphatase and UDPglucuronyl transferase activities, but they did not closely correlate with the alterations in acyl-CoA:cholesterol acyltransferase activity. Electron spin resonance measurements with the 5-nitroxystearate probe indicated that microsomal lipid ordering was reduced to a roughly similar extent by dioleoyl- or by dilinoleoylphosphatidylcholine enrichment. Since these enrichments produce widely different effects on acyl-CoA:cholesterol acyltransferase activity, changes in bulk membrane lipid fluidity cannot be the only factor responsible for phospholipid fatty acid compositional effect on acyl-CoA:cholesterol acyltransferase. The present results are more consistent with a modulation resulting from either changes in the lipid microenvironment of acyl-CoA:cholesterol acyltransferase or a direct interaction between specific phosphatidylcholine fatty acyl groups and acyl-CoA:cholesterol acyltransferase.     

Apolipoprotein A-I: structure-function relationships

The inverse relationship between high density lipoprotein (HDL) plasma levels and coronary heart disease has been attributed to the role that HDL and its major constituent, apolipoprotein A-I (apoA-I), play in reverse cholesterol transport (RCT). The efficiency of RCT depends on the specific ability of apoA-I to promote cellular cholesterol efflux, bind lipids, activate lecithin:cholesterol acyltransferase (LCAT), and form mature HDL that interact with specific receptors and lipid transfer proteins. From the intensive analysis of apoA-I secondary structure has emerged our current understanding of its different classes of amphipathic alpha-helices, which control lipid-binding specificity. The main challenge now is to define apoA-I tertiary structure in its lipid-free and lipid-bound forms. Two models are considered for discoidal lipoproteins formed by association of two apoA-I with phospholipids. In the first or picket fence model, each apoA-I wraps around the disc with antiparallel adjacent alpha-helices and with little intermolecular interactions. In the second or belt model, two antiparallel apoA-I are paired by their C-terminal alpha-helices, wrap around the lipoprotein, and are stabilized by multiple intermolecular interactions. While recent evidence supports the belt model, other models, including hybrid models, cannot be excluded. ApoA-I alpha-helices control lipid binding and association with varying levels of lipids. The N-terminal helix 44-65 and the C-terminal helix 210-241 are recognized as important for the initial association with lipids. In the central domain, helix 100-121 and, to a lesser extent, helix 122-143, are also very important for lipid binding and the formation of mature HDL, whereas helices between residues 144 and 186 contribute little. The LCAT activation domain has now been clearly assigned to helix 144-165 with secondary contribution by helix 166-186. The lower lipid binding affinity of the region 144-186 may be important to the activation mechanism allowing displacement of these apoA-I helices by LCAT and presentation of the lipid substrates. No specific sequence has been found that affects diffusional efflux to lipid-bound apoA-I. In contrast, the C-terminal helices, known to be important for lipid binding and maintenance of HDL in circulation, are also involved in the interaction of lipid-free apoA-I with macrophages and specific lipid efflux. While much progress has been made, other aspects of apoA-I structure-function relationships still need to be studied, particularly its lipoprotein topology and its interaction with other enzymes, lipid transfer proteins and receptors important for HDL metabolism.     

A novel lecithin-cholesterol acyltransferase antioxidant activity prevents the formation of oxidized lipids during lipoprotein oxidation.

Recent investigations suggest that high-density lipoprotein (HDL) may play an anti-atherogenic role as an antioxidant and inhibit the oxidative modification of low-density lipoprotein (LDL). The antioxidant activity of HDL has been proposed to be associated with several HDL-bound proteins. We have purified one HDL-associated protein, lecithin:cholesterol acyltransferase (LCAT), to apparent homogeneity and have found that LCAT is not only capable of esterifying cholesterol in the plasma, but can also prevent the accumulation of oxidized lipids in LDL. Addition of pure human LCAT to LDL or palmitoyl-linoleoyl phosphatidylcholine/sodium cholate (PLPC) micelles inhibits the oxidation-dependent accumulation of both conjugated dienes and lipid hydroperoxides. LCAT also inhibits the increase of net negative charge that occurs during oxidation of LDL. LCAT has the ability to prevent spontaneous oxidation and Cu2+ and soybean lipoxygenase-catalyzed oxidation of lipids. The antioxidant activity of LCAT appears to be enzymatic, since the enzyme is active for up to 10 h in the presence of mild free-radical generators. The catalytic serine, residue 181, may mediate this activity and act as a reusable proton donor. Chemical modification of the active serine residue with diisopropylfluorophosphate completely inhibits the ability of LCAT to prevent lipid oxidation. Thus, in addition to its well-characterized phospholipase and acyltransferase activities, LCAT can also act as an antioxidant and prevent the accumulation of oxidized lipid in plasma lipoproteins.     

Synthetic substrates of lecithin: cholesterol acyltransferase

Investigation of the substrate specificity of lecithin: cholesterol acyltransferase has been greatly aided by the use of synthetic particles containing the molecular lipid substrates and the apolipoprotein activators of the enzyme. These synthetic particles, in vesicle or disc-like micelle form, are described in some detail noting their preparation, properties, advantages, and limitations as substrates for lecithin:cholesterol acyltransferase. The reactions of the enzyme with the synthetic particles are reviewed in terms of acyl donor and acceptor specificity, activation by apolipoproteins, effects of various inhibitors, and the kinetics of the reaction.     

Acyl chain and headgroup specificity of human plasma lecithin:cholesterol acyltransferase. Separation of matrix and molecular specificities

To determine how substrate fluidity and molecular structure independently regulate cholesteryl ester formation, the substrate specificity of lecithin:cholesterol acyltransferase with respect to a number of model reassembled high density lipoproteins (R-HDLs) is reported. The R-HDLs are composed of 1 mol % apolipoprotein A-I, 89 mol % of sphingomyelin or a nonhydrolyzable diether analog of phosphatidylcholine (PC) plus 10 mol % of test lipids that are potential acyl donors; a trace of [3H]cholesterol, which permits quantification of cholesteryl ester formation is also included. With respect to the lipid class of the acyl donor, the rate of ester formation decreases in the order phosphatidylethanolamine greater than phosphatidylcholine greater than N,N,-dimethylphosphatidylethanolamine greater than phosphatidylglycerol - phosphatidic acid greater than phosphatidylserine greater than dipalmitin greater than tripalmitin. Within an R-HDL composed of 90% PC ether or sphingomyelin, the relative rates of ester formation are greatest for dipalmitoyl and dimyristoyl PC, with distearoyl PC being almost unreactive; in a solid lipid environment, the rate with respect to unsaturation of the PC is greatest for oleate. In a fluid lipid environment, all unsaturated PCs were utilized nearly equally. All lipids tested were most reactive within an R-HDL composed of an unsaturated PC ether and least reactive within an R-HDL composed mostly of sphingomyelin. These results suggest that the rates of ester formation by lecithin:cholesterol acyltransferase are separate functions of the identity and the microscopic environment of the acyl donor. This is the first example of the use of diether analogs for the separation of the effects of macromolecular and molecular structure on the specificity of lecithin:cholesterol acyltransferase.     

Photo-induced dimerization of anthracene phospholipids for the study of the lateral distribution of lipids in membranes

The biosynthesis of linoleic acid has been investigated, using oleoyl-CoA as a substrate, in microsomal preparations from young leaves of Pisum sativum. Oleoyl moieties from oleoyl-CoA were preferentially acylated to lysophosphatidylcholine by an acyltransferase to produce an oleoylglycerophosphocholine. Kinetic data are presented which argue for a direct desaturation of the oleoyl moieties of this oleoyl glycerophosphocholine to linoleoyl moieties. There was no evidence of a subsequent acyltransfer of linoleoyl moieties either to form thioesters or oxygen esters in other complex lipids. The kinetics were also consistent with a functional coupling of the lysophosphatidylcholine acyltransferase with the oleate desaturase. There was little exchange of the oleoyl glycerophosphocholine from the bulk membrane lipid with that newly synthesised by the lysophosphatidylcholine acyltransferase. Rather, the newly synthesised oleoylglycerophosphocholine seemed to be directly channelled to the vicinity of the desaturase. The results are discussed in the context of 'metabolite channelling'. The consequences for desaturase activity and its regulation are also examined.     

Isolation and characterization of Chinese hamster ovary cell mutants deficient in acyl-coenzyme A:cholesterol acyltransferase activity

A protocol has been developed for isolating cholesterol ester-deficient cells from the Chinese hamster ovary cell clone 25-RA. This cell line previously was shown to be partially resistant to suppression of cholesterogenic enzyme activities by 25-hydroxycholesterol and to accumulate a large amount of intracellular cholesterol ester when grown in medium containing 10% fetal calf serum (Chang, T. Y., and Limanek, J. S. (1980) J. Biol. Chem. 255, 7787-7795). The higher cholesterol ester content of 25-RA is due to an increase in the rate of cholesterol biosynthesis and low density lipoprotein receptor activity compared to wild-type Chinese hamster ovary cells, and not due to an abnormal acyl-CoA:cholesterol acyltransferase enzyme. The procedure to isolate cholesterol ester-deficient mutants utilizes amphotericin B, a polyene antibiotic known to bind to cholesterol and to form pore complexes in membranes. After incubation in cholesterol-free medium plus an inhibitor of endogenous cholesterol biosynthesis, 25-RA cells were found to be 50-500 times more sensitive to amphotericin B killing than were mutant cells containing reduced amounts of cholesterol ester. Twelve amphotericin B-resistant mutants were isolated which retained the 25-hydroxycholesterol-resistant phenotype. These mutants did not exhibit the perinuclear lipid droplets characteristic of 25-RA cells, and lipid analysis revealed a large (up to 40-fold) reduction in cellular cholesterol ester. The acyl-CoA:cholesterol acyltransferase activities of these cholesterol ester-deficient mutants were markedly lower than 25-RA when assayed in intact cells or in an in vitro reconstitution assay. The tightest mutant characterized, AC29, was found to have less than 1% of the parental acyl-CoA:cholesterol acyltransferase activity. These mutants all have reduced rates of sterol synthesis and lower low density lipoprotein receptor activity compared to 25-RA, probably as a consequence of their reduced enzyme activities. Cell fusion experiments revealed that the phenotypes of all the mutants examined are not dominant and that the mutants all belong to the same complementation group. We conclude that these mutants contain a lesion in the gene encoding acyl-CoA:cholesterol acyltransferase or in a gene encoding a factor needed for enzyme production.     

Identification and characterization of DGA2, an acyltransferase of the DGAT1 acyl-CoA:diacylglycerol acyltransferase family in the oleaginous yeast Yarrowia lipolytica. New insights into the storage lipid metabolism of oleaginous yeasts

Triacylglycerols (TAG) and steryl esters (SE) are the principal storage lipids in all eukaryotic cells. In yeasts, these storage lipids accumulate within special organelles known as lipid bodies (LB). In the lipid accumulation-oriented metabolism of the oleaginous yeast Yarrowia lipolytica, storage lipids are mostly found in the form of TAG, and only small amounts of SE accumulate. We report here the identification of a new DAG acyltransferase gene, DGA2, homologous to the ARE genes of Saccharomyces cerevisiae. This gene encodes a member of the type 1 acyl-CoA:diacylglycerol acyltransferase family (DGAT1), which has not previously been identified in yeasts, but is commonly found in mammals and plants. Unlike the Are proteins in S. cerevisiae, Dga2p makes a major contribution to TAG synthesis via an acyl-CoA-dependent mechanism and is not involved in SE synthesis. This enzyme appears to affect the size and morphology of LB, suggesting a direct role of storage lipid proteins in LB formation. We report that the Are1p of Y. lipolytica was essential for sterol esterification, as deletion of the encoding gene (ARE1) completely abolished SE synthesis. Unlike its homologs in yeasts, YlARE1 has no DAG acyltransferase activity. We also reconsider the role and function of all four acyltransferase enzymes involved in the final step of neutral lipid synthesis in this oleaginous yeast.     

Helicobacter pylori lipids can form ordered membrane domains (rafts)

Ordered lipid domains (rafts) are generally considered to be features of eukaryotic cells, but ordered lipid domains formed by cholesterol lipids have been identified in bacteria from the genus Borrelia, and similar cholesterol lipids exist in the bacterium Helicobacter pylori. To determine whether H. pylori lipids could form ordered membrane domains, we investigated domain formation in aqueous dispersions of H. pylori whole lipid extracts, individual H. pylori lipids, or defined mixtures of H. pylori lipids and other membrane-forming lipids. DPH (1,6-diphenyl-1,3,5-hexatriene) anisotropy measurements were used to assay membrane order and FRET (Förster resonance energy transfer) was used to detect the presence of co-existing ordered and disordered domains. We found that H. pylori membrane lipid extracts spontaneously formed lipid domains. Domain formation was more stable when lipids were extracted from H. pylori cells grown in the presence of cholesterol. Certain isolated H. pylori lipids (by themselves or when mixed with other lipids) also had the ability to form ordered domains. To be specific, H. pylori cholesteryl-6-O-tetradecanoyl-α-D-glucopyranoside (CAG) and cholesterol-6-O-phosphatidyl-α-D-glucopyranoside (CPG) had the ability to form and/or stabilize ordered domain formation, while H. pylori phosphatidylethanolamine did not, behaving similarly to unsaturated phosphatidylethanolamines. We conclude that specific H. pylori cholesterol lipids have a marked ability to form ordered lipid domains.     

The activity of 3-hydroxy-3-methylglutaryl-CoA reductase and acyl-CoA: cholesterol acyltransferase in hepatic microsomes from male, female and pregnant rats. The effect of cholestyramine treatment and the relationship of enzyme activity to microsomal lipid composition

The relationship of microsomal cholesterol and phospholipid fatty acid composition to the activities of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase and acyl-CoA: cholesterol acyltransferase was investigated in male, female virgin and pregnant rats when hepatic cholesterogenesis was stimulated by cholestyramine. Cholestyramine increased HMG-CoA reductase activity in both sexes but had no effect on microsomal free cholesterol level or acyl-CoA: cholesterol acyltransferase activity. The data suggest that during cholestyramine treatment high rates of bile acid synthesis are supported by preferential channelling of cholesterol into this pathway, whilst the substrate pool and activity of acyl-CoA:cholesterol acyltransferase are maintained unaltered. The lack of a consistent relationship among enzyme activities and microsomal lipid composition infers that HMG-CoA reductase and acyl-CoA:cholesterol acyltransferase are regulated in vivo by independent mechanisms which are unlikely to involve modulation by the physical properties of the microsomal lipid.     

种子萌发过程中贮藏油脂的动员

对植物种子萌发过程中贮藏油脂动员的研究进展进行了综述。不同种子的贮藏油脂的降解途径不同。目前提出有3条途径:传统的脂酶直接水解途径;新近发现的酰基-CoA-二酯酰甘油酰基转移酶途径和脂氧合酶(LOX)途径。前两条途径不依赖于LOX。这3条途径可能在贮藏油脂动员过程中是并存的,但目前尚不知道在种子萌发过程中油脂降解是以那一条降解途径为主,以及不同的种之间是否存在差异。此外,3条降解途径目前都缺乏分子生物学的直接证据。     

Effect of methotrexate on long-chain fatty acid metabolism in liver of rats fed a standard or a defined, choline-deficient diet

The effect of methotrexate on lipids in serum and liver and key enzymes involved in esterification and oxidation of long-chain fatty acids were investigated in rats fed a standard diet and a defined choline-deficient diet. Hepatic metabolism of long-chain fatty acids were also studied in rats fed the defined diet with or without choline. When methotrexate was administered to the rats fed the standard diet there was a slight increase in hepatic lipids and a moderate reduction in the serum level. The palmitoyl-CoA synthetase activity and the microsomal glycerophosphate acyltransferase activity in the liver of rats were increased by methotrexate. The data are consistent with those where the liver may fail to transfer the newly formed triacylglycerols into the plasma with a resultant increase in liver triacylglycerol content and a decrease in serum lipid levels. Fatty liver of methotrexate-exposed rats can not be attributed simply to a reduction of fatty acid oxidation as the carnitine palmitoyltransferase activity was increased. The methotrexate response in the rats fed the defined choline-deficient diet was different. There was a reduction in both serum and hepatic triacylglycerol and the glycerophosphate acyltransferase and palmitoyl-CoA synthetase activities. The carnitine palmitoyltransferase activity was unchanged. Hepatomegaly and increased hepatic fat content, but decreased serum triacylglycerol, total cholesterol and HDL cholesterol were found to be related to the development of choline deficiency as the pleiotropic responses were almost fully prevented by addition of choline to the choline-deficient diet. Addition of choline to the choline-deficient diet normalized the total palmitoyl-CoA synthetase and carnitine palmitoyltransferase activities. In contrast to methotrexate exposure, choline deficiency increased the mitochondrial glycerophosphate acyltransferase activity. The data are consistent with those of where fatty liver induction of choline deficiency may be related to an enhanced esterification of long-chain fatty acids concomitant with a reduction of their oxidation.     

Host cell lipids control cholesteryl ester synthesis and storage in intracellular Toxoplasma

The intracellular protozoan Toxoplasma gondii lacks a de novo mechanism for cholesterol synthesis and therefore must scavenge this essential lipid from the host environment. In this study, we demonstrated that T. gondii diverts cholesterol from low-density lipoproteins for cholesteryl ester synthesis and storage in lipid bodies. We identified and characterized two isoforms of acyl-CoA:cholesterol acyltransferase (ACAT)-related enzymes, designated TgACAT1alpha and TgACAT1beta in T. gondii. Both proteins are coexpressed in the parasite, localized to the endoplasmic reticulum and participate in cholesteryl ester synthesis. In contrast to mammalian ACAT, TgACAT1alpha and TgACAT1beta preferentially incorporate palmitate into cholesteryl esters and present a broad sterol substrate affinity. Mammalian ACAT-deficient cells transfected with either TgACAT1alpha or TgACAT1beta are restored in their capability of cholesterol esterification. TgACAT1alpha produces steryl esters and forms lipid bodies after transformation in a Saccharomyces cerevisiae mutant strain lacking neutral lipids. In addition to their role as ACAT substrates, host fatty acids and low-density lipoproteins directly serve as Toxoplasma ACAT activators by stimulating cholesteryl ester synthesis and lipid droplet biogenesis. Free fatty acids significantly increase TgACAT1alpha mRNA levels. Selected cholesterol esterification inhibitors impair parasite growth by rapid disruption of plasma membrane. Altogether, these studies indicate that host lipids govern neutral lipid synthesis in Toxoplasma and that interference with mechanisms of host lipid storage is detrimental to parasite survival in mammalian cells.     

The modulation of membrane fluidity by hydrogenation processes. II. Homogeneous catalysis and model biomembranes

A homogeneous catalyst, chlorotris (triphenylphosphine) rhodium (I) has been incorporated into model biomembrane structures in the form of lipid bilayer dispersions in water. This enables the hydrogenation of the double bonds of the unsaturated lipids within the bilayers to be accomplished. To decide the optimum conditions for efficient hydrogenation the reaction conditions have been varied. The effect of catalyst concentration, hydrogen gas pressure and lipid composition (with and without cholesterol) have all been studied. The partition of the catalyst into the lipid medium was checked by rhodium analysis. The results show that an increase of catalyst concentration or an increase of hydrogen gas pressure leads to increasing rates of hydrogenation. Successful hydrogenation was accomplished with different types of lipid dispersions (mitochondrial, microsomal and erythrocyte lipids). A selectivity of the homogeneous hydrogenation process is indicated. The polyunsaturated fatty acyl residues are hydrogenated at an earlier stage and at a faster rate than the monoenoic acids. Furthermore, an increase in the proportion of cholesterol to lipid within the bilayer structures causes a progressive decrease in the rate of hydrogenation. The fluidity of the lipid bilayers can be altered to such an extent by the hydrogenation process that new sharp endotherms corresponding to the order-disorder transition of saturated lipids occur at temperatures as high as 319 K. Some potential uses of hydrogenation for the modulation of cell membrane fluidity are discussed as well as the design of new types of catalyst molecules.     

Domain coupling in asymmetric lipid bilayers

Biological membranes are heterogeneous assemblies of lipids, proteins, and cholesterol that are organized as asymmetric bimolecular leaflets of lipids with embedded proteins. Modulated by the concentration of cholesterol lipids and proteins may segregate into two or more liquid phases with different physical properties that can coexist in the same membrane. In this review, we summarize recent advances on how this situation can be recreated in a supported bilayer format and how this system has been used to demonstrate the induction of ordered lipid domains in lipid compositions that are typical for the inner leaflet by lipid compositions that are typical for the outer leaflet of mammalian plasma membranes. Proteins are shown to differentially target such induced inner leaflet domains.     

Self-adaptive modification of red-cell membrane lipids in lecithin: Cholesterol acyltransferase deficiency. Lipid analysis and spin labeling

In a patient with lecithin: cholesterol acyltransferase deficiency, free cholesterol was markedly increased, and esterified cholesterol was diminished. In the patient's plasma, an increase in phosphatidylcholine (PC) and a decrease in sphingomyelin were observed. Concomitantly, an increase in a shorter acyl chain 16:0 was noted in PC, sphingomyelin and phosphatidylethanolamine (PE). In contrast to these results, longer chains such as 22:0 and 24:0 were decreased, especially in sphingomyelin. Unsaturated double bonds such as 18:1 was also increased in PC and PE. In the red-cell membrane lipids, the increase in free cholesterol was counteracted by an increase in PC and by a decrease in sphingomyelin and PE, reflecting changes in the patient's plasma lipids. Increased 16:0 (in PC) and decreased 18:0 and 24:0 were observed. The increased plasma free cholesterol due to metabolic defect (lecithin:cholesterol acyltransferase deficiency) led to decreased red-cell membrane fluidity. This effect appeared to be counteracted by changing phospholipid composition (increased PC and decreased sphingomyelin and PE), by increasing shorter chains (16:0), by decreasing longer chains (18:0 and 24:0) and by increasing unsaturated double bonds (18:2). These results can be interpreted as a self-adaptive modification of lecithin:cholesterol acyltransferase deficiency-induced red-cell membrane abnormalities, to maintain normal membrane fluidity. This speculation was supported by the ESR spin-label studies on the patient's membrane lipids. The normal order parameters in intact red cells and in total lipid liposomes were decreased if cholesterol-depleted membrane liposomes were prepared. Thus, the hardening effect of cholesterol appeared to be counteracted by the softening effects described above. Overall membrane fluidity in intact red cells of the lecithin:cholesterol acyltransferase-deficient patient was maintained normally, judged by order parameters in ESR spin-label studies.     

Domain coupling in asymmetric lipid bilayers

Biological membranes are heterogeneous assemblies of lipids, proteins, and cholesterol that are organized as asymmetric bimolecular leaflets of lipids with embedded proteins. Modulated by the concentration of cholesterol lipids and proteins may segregate into two or more liquid phases with different physical properties that can coexist in the same membrane. In this review, we summarize recent advances on how this situation can be recreated in a supported bilayer format and how this system has been used to demonstrate the induction of ordered lipid domains in lipid compositions that are typical for the inner leaflet by lipid compositions that are typical for the outer leaflet of mammalian plasma membranes. Proteins are shown to differentially target such induced inner leaflet domains.     

Serum and liver lipid composition and lecithin: cholesterol acyltransferase in horses, Equus caballus.

1. The lipid composition of serum and liver and some properties of serum lecithin: cholesterol acyltransferase of the horse were investigated. 2. Phospholipids and cholesterol were the major components of serum lipids and the concentration of triglyceride was considerably low. The concentration of liver lipids was comparable with that of other mammals. 3. Fatty acid composition of serum cholesterol ester resembled that of the 2-position of lecithin, except palmitic acid. 4. The activity of serum cholesterol esterifying enzyme was found to be 0.03-0.09 mumol/hr per ml. There was an equimolar decrease in free cholesterol and lecithin during incubation, and changes in unsaturated fatty acids in these two components were in good agreement. 5. Cholesterol esterification was reversibly inhibited by 5,5'-dithiobis-(2-nitrobenzoic acid). The acyl-transferase had a specificity for linoleic acid.     

Thematic review series: skin lipids. The role of epidermal lipids in cutaneous permeability barrier homeostasis

The permeability barrier is required for terrestrial life and is localized to the stratum corneum, where extracellular lipid membranes inhibit water movement. The lipids that constitute the extracellular matrix have a unique composition and are 50% ceramides, 25% cholesterol, and 15% free fatty acids. Essential fatty acid deficiency results in abnormalities in stratum corneum structure function. The lipids are delivered to the extracellular space by the secretion of lamellar bodies, which contain phospholipids, glucosylceramides, sphingomyelin, cholesterol, and enzymes. In the extracellular space, the lamellar body lipids are metabolized by enzymes to the lipids that form the lamellar membranes. The lipids contained in the lamellar bodies are derived from both epidermal lipid synthesis and extracutaneous sources. Inhibition of cholesterol, fatty acid, ceramide, or glucosylceramide synthesis adversely affects lamellar body formation, thereby impairing barrier homeostasis. Studies have further shown that the elongation and desaturation of fatty acids is also required for barrier homeostasis. The mechanisms that mediate the uptake of extracutaneous lipids by the epidermis are unknown, but keratinocytes express LDL and scavenger receptor class B type 1, fatty acid transport proteins, and CD36. Topical application of physiologic lipids can improve permeability barrier homeostasis and has been useful in the treatment of cutaneous disorders.