High performance thin-layer chromatography and densitometry of synaptic plasma membrane lipids

Previously, it has been shown that phospholipids, cholesterol, and glycolipids could be quantitated using the same high performance thin-layer chromatography (HPTLC) method. Here we examined that method in terms of linearity of standards in the nanogram range, recovery of nonacidic and acidic lipids after Sephadex column chromatography, and quantitation of lipids in mouse synaptic plasma membranes (SPM) where lipid content is low. Nonacidic and acidic fractions were separated by Sephadex column chromatography, applied to plates using contact spotting, chromatographed, visualized with cupric acetate, and quantitated using in situ densitometry. Recovery of nonacidic and acidic fractions off the columns was determined with radiolabeled phospholipids. Standards for each lipid class were linear in the nanogram range. Quantitation of SPM lipid classes could be made with as little as 1.5 micrograms of total lipid. Recovery of the nonacidic fraction after Sephadex column chromatography was approximately 100% whereas the acidic fraction was approximately 91%. Phospholipids, cholesterol, and glycolipids could be determined in nanogram amounts using the same method. This method is an efficient method for examining different lipid classes and in samples where lipid content is low.     

Plasmodium falciparum and P. knowlesi: initial identification and characterization of malaria synthesized glycolipids

This is the first report establishing the existence of glycolipids synthesized by plasmodia, in particular Plasmodium falciparum. Trophozoites, schizonts, gametocytes, and gametes were metabolically labeled in vitro with [3H]glucosamine, [3H]galactose, [3H]glucose, [3H]mannose, [3H]fucose, [32P]inorganic phosphate, or [35S]sulfate, and total lipid extracts analyzed by high-performance thin-layer chromatography and autoradiography or fluorography. Parasites incorporated [3H]monosaccharides into distinctly different series of molecules previously undescribed. Three properties of [3H]glucosamine labeled molecules indicate they are glycolipids. First, labeled molecules have lipid solubility properties. Second, mobility on thin-layer chromatography was characteristic of glycolipids. Third, following acid hydrolysis, [3H]glucosamine was recovered from a total lipid extract of labeled parasites demonstrating that glucosamine is a constituent of some of these lipid molecules. Most of these glycolipids are neutral and alkali labile. The majority of these glycolipids differs from several synthesized phospholipids. None of these glycolipids was sulfated. Plasmodial glycolipid synthesis occurs concomitantly with glycoprotein synthesis, and both increase during schizogony. Many of these glycolipids appear to be identical among three strains of P. falciparum and between two species, P. falciparum and P. knowlesi. In contrast, there are stage specific differences in glycolipid synthesis among rings, schizonts, gametocytes, and a mixture of gametes plus zygotes of P. falciparum, examples of both erythrocytic and vector forms of the parasite.     

Purification of Forssman and human blood group A glycolipids by affinity chromatography on immobilized Helix pomatia lectin

A method for the affinity purification of intact glycolipids having nonreducing terminal alpha 1-3 linked N-acetylgalatosamine residues has been developed. This technique relies on the retention of the carbohydrate-binding specificity of immobilized Helix pomatia lectin in aqueous solutions of tetrahydrofuran. Both Forssman glycolipid and a mouse blood group A-active hexaosylceramide were bound by columns of the lectin equilibrated in a solvent containing 95% tetrahydrofuran and 5% water. After application of a step gradient of increasing water content up to 50%, the specifically bound glycolipids were eluted in solvent containing N-acetylgalactosamine. The Forssman and A-active glycolipids were similarly purified in a single chromatographic step from total lipid extracts of sheep and human type A erythrocyte stroma, respectively. Nonspecifically bound lipids and glycolipids were eluted from this column by simply increasing the water content of the eluting buffer. The extension of this method to other carbohydrate-binding proteins including lectins and monoclonal antibodies may provide a rapid purification of glycolipids based on their carbohydrate structures.     

Microvillus membrane vesicles from pig small intestine. Purity and lipid composition

Microvillus membrane vesicles from pig small intestine were isolated by a method based on hypotonic lysis, Mg2+-aggregation of contaminants and differential centrifugation. The purity of the membrane vesicles were established by measuring the activity of marker enzymes and the RNA and DNA content. The membranes were found free of contamination by other subcellular membrane fragments, except for a minor contamination with basolateral plasma membranes. The lipid composition was established and, based on weight percentage, the membrane contained neutral lipids, phospholipids, neutral glycolipids and gangliosides in the weight ratio of 18 : 50 : 29 : 2%. The amount of individual phospholipids and glycolipids were quantitated. Phosphatidylethanolamine, -choline, -serine, -inositol and sphingomyelin made up 17, 17, 6, 5 and 5%, respectively of the total lipid. The major glycolipids were two monohexosylceramides containing glucose and galactose as the carbohydrate component, a dihexosylceramide containing galactose as the only carbohydrate component and two pentahexosylceramides containing fucose, galactose, glucose and hexosamine (either N-acetylglucosamine or N-acetylgalactosamine) in the molar ratio of 1 : 2 : 1 : 1.     

Structural analysis of glycolipids from Borrelia burgdorferi

In this study the lipids of Borrelia burgdorferi, the causative agent of Lyme disease, were analyzed. Lipids comprise about 25-30% of the cell dry weight. The lipid fraction could be separated by HPTLC into 11 components. Staining of these components revealed two glycolipids and two phospholipids. The glycolipids represented about 50% of the total lipids and comprised only galactose as monosaccharide constituents. By means of mass spectrometric and gas chromatographic analysis both glycolipids could be identified as alpha-galactosyl-diacylglycerolipids with different fatty acid compositions. The phospholipids were identified as phosphatidylcholine and phosphatidylglycerol. Immunoassays with sera from patients with Lyme disease showed antibody reactivity only to the glycolipids, which was present in all stages of the disease. Other lipid components seemed to be non-immunogenic in Lyme disease. The glycolipids of B. burgdorferi may be, thus, considered promising candidates for diagnosis and possibly also for vaccination.     

ESI-MS MS方法检测拟南芥膜脂分子对机械伤害的响应

利用一种灵敏的、基于ESI-MS􊄯MS ( electrospray ionization tandem mass spectrometry) 的脂类组学方法,测定了机械伤害诱导的拟南芥6 种磷 (phospholipids) 、2 种糖脂(glycolipids) 、3 种溶血磷脂( lysophospholipids)和约120 种脂类分子的变化, 探索了膜脂响应机械伤害的基本趋势。结果表明, 机械伤害后磷脂酸( phosphatidic acid , PA) 和3 种溶血磷脂显著升高, 而叶绿体膜上的糖脂减少; 在测量的1 小时范围内, 不同脂类水解产生的磷脂酸分子的增加速度和强度不同, 反映出它们经历了不同的生化过程。具体表现为:(1 ) 叶绿体膜脂磷脂酰甘油(phosphatidylglycero , PG) 分子34∶4 PG 水解的产物磷脂酸分子34∶4 PA 的积累速度明显慢于其它磷脂酸分子; (2) 磷脂酸分子34∶6 PA 仅有少量的积累, 其可能是由叶绿体膜脂单半乳糖二酰甘油(monogalactosyldiacylglycerol, MGDG) 。分子34∶6 MGDG 和双半乳糖二酰甘油( digalactosyldiacylglycerol, DGDG) 分子34∶6 DGDG 水解产生, 然而这两种糖脂含量明显下降, 说明它们有可能还参与了其它的反应。脂类的摩尔百分组成没有剧烈的变化。     

Characterization of membrane components of the flask-shaped mycoplasma Mycoplasma mobile

The cell membrane of Mycoplasma mobile was isolated by either ultrasonic or French press treatment of intact cells. The membrane fraction contained all of the cellular lipids, but only one-third of cellular proteins and had a density of 1.14 g ml-1. The soluble fraction contained the NADH dehydrogenase activity of the cells, as well as a protein with an apparent molecular mass of 55 kDa that was phosphorylated in the presence of ATP. Lipid analyses of M. mobile membranes revealed that membrane lipid could be labelled by radioactive glycerol, oleate and to a much higher extent by palmitate but not by acetic acid. The membrane lipid fraction was composed of 54% neutral and 46% polar lipid. The major constituents of the neutral lipid fraction were free fatty acid, free cholesterol and cholesterol esters (45, 25 and 20%, respectively, of total neutral lipid fraction). The free cholesterol count was 13% (w/w) of total membrane lipids with a cholesterol:phospholipid molar ratio of about 0.9. Among the polar lipids, both phospho- and glycolipids were detected. The phospholipid fraction consisted of a major de novo-synthesized phosphatidylglycerol (approximately 63% of total phospholipids), plus exogenous phosphatidylcholine and sphingomyelin incorporated in an unchanged form from the growth medium. The glycolipid fraction was dominated by a single glycolipid (approximately 90% of total glycolipids) that was preferentially labelled by palmitic acid and showed a very high saturated:unsaturated fatty acids ratio.     

Quantitation of galactocerebrosides and sulfatides by galactose oxidase and sodium borotritide

A method has been developed for the quantitation of galactocerebrosides using galactose oxidase and sodium borotritide that is highly specific for terminal galactose or galactosamine containing glycolipids. Prior separation of galactocerebrosides from other lipids is unnecessary and nanomole quantities of galactocerebrosides can be reliably estimated within 6–7 h. This method can be adapted with slight modification for quantitation of sulfatides in total lipid extract.     

The isolation and partial characterization of the glycolipids of BP8/C3H ascites-sarcoma cells

1. The total lipid was extracted from BP8/C3H ascites-sarcoma cells with acetone, light petroleum, pyridine and chloroform–methanol successively. Each extract was treated with mild alkali. The alkali-stable lipids from the pyridine and chloroform–methanol extracts, which included the glycolipids, were fractionated on silicic acid and silica gel G columns. 2. The total yield of glycolipid was about 60 mg./100 g. dry wt. of tumour cells, about 0·4% of the total lipid. Four classes of glycolipid were isolated and characterized as ceramide monohexoside (G1), ceramide dihexoside (G2), ceramide trihexoside (G3) and ceramide hexosaminyltrihexoside (G4). 3. G1, G2, G3 and G4 constituted 55, 21, 9 and 15% of the total glycolipid respectively. 4. G1 was a mixture of ceramide glucoside (70%) and ceramide galactoside. 5. The general structures of the oligosaccharide moieties of G2, G3 and G4 were elucidated by partial acid hydrolysis of the glycolipids with water-soluble polystyrenesulphonic acid. G2 was mostly ceramidelactoside with about 10% of ceramide galactosylgalactoside. G3 and G4 were probably a ceramide digalactosylglucoside and a ceramide N-acetylgalactosaminylgalactosylgalactosylglucoside respectively. 6. The fatty acid compositions of the glycolipids were very similar; lignoceric acid and nervonic acid were the major components and all contained monohydroxy acids in proportions varying from 10 to 25% of the total acids.     

Separation of neutral glycosphingolipids and sulfatides by thin-layer chromatography

Two one-dimensional systems for separation of glycolipids from total lipid extracts of tissues by thin-layer chromatography are described. System I used, as adsorbent, an alkaline mixture of silica gel without CaSO(4) binder (75%) and magnesium silicate (25%), and the lipids were "developed" with three successive solvent mixtures. The separated compounds (from the fastest to the slowest moving) were: ceramide, ceramide monohexosides, sulfatides, ceramide dihexosides, psychosine, ceramide trihexosides, and ceramide N-acetylhexosamine trihexosides. In system II a two-step development was used on an adsorbent consisting of silica gel without CaSO(4) binder (80%) and magnesium silicate (20%). The separated compounds were: ceramides, ceramide monohexosides, and ceramide dihexosides. Psychosine and sulfatides as well as ceramide trihexosides and ceramide N-acetylhexosamine trihexosides were not separated. In both systems all neutral lipids moved to the very top of the chromatogram and phospholipids stayed at the origin. Application of systems I and II for separation of glycolipids was demonstrated on total lipid extracts from animal tissues.     

Microvillus membrane vesicles from pig small intestine Purity and lipid composition

Microvillus membrane vesicles from pig small intestine were isolated by a method based on hypotonic lysis, Mg²⁺ aggregation of contaminants and differential centrifugation. The purity of the membrane vesicles were established by measuring the activity of marker enzymes and the RNA and DNA content. The membranes were found free of contamination by other subcellular membrane fragments, except for a minor contamination with basolateral plasma membranes. The lipid composition was established and, based on weight percentage, the membrane contained neutral lipids, phospholipids, neutral glycolipids and gangliosides in the weight ratio of 18:50:29:2%. The amount of individual phospholipids and glycolipids were quantitated. Phosphatidylethanolamine, -choline, -serine, -inositol and sphingomyelin made up 17,17,6,5 and 5%, respectively of the total lipid. The major glycolipids were two monohexosylceramides containing glucose and galactose as the carbohydrate component, a dihexosylceramide containing galactose as the only carbohydrate component and two pentahexosylceramides containing fucose, galactose, glucose and hexosamine (either $\text{N-}\text{acetylglucosamine}$ or $\text{N-}\text{acetylgalactosamine}$) in the molar ratio of 1:2:1:1.     

Raft trafficking of AB5 subunit bacterial toxins

Cholera and the related AB(5)-subunit toxins co-opt plasma membrane (PM) glycolipids to move retrograde into the endoplasmic reticulum (ER) of the host cell where a portion of the toxin is retro-translocated to the cytosol to induce disease. Only glycolipids that associate strongly with detergent insoluble membrane microdomains can sort the toxins backwards from PM to ER. The way certain lipids and proteins are clustered in the plane of the membrane to form lipid rafts likely explains how the glycolipids can function as sorting motifs for the toxins.     

Toxin A from Clostridium difficile binds to rabbit erythrocyte glycolipids with terminal Gal alpha 1-3Gal beta 1-4GlcNAc sequences

The binding of Toxin A isolated from Clostridium difficile to rabbit erythrocyte glycolipids has been studied. Total lipid extracts from rabbit erythrocytes were subjected to thin-layer chromatography and toxin-binding glycolipids detected by using 125I-labeled Toxin A in a direct binding overlay technique. Two major and several minor toxin-binding glycolipids were detected in rabbit erythrocytes by this method. The results of structural analyses of the major toxin-binding glycolipids were consistent with a pentasaccharide-ceramide (Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc-Cer) and a branched decasaccharide-ceramide (Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3[Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-6]Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc-Cer) previously identified as the two most abundant glycolipids in rabbit erythrocytes. 125I-Toxin A binding to these glycolipids could be inhibited by bovine thyroglobulin, monospecific antiserum to the toxin, or by treatment of the glycolipids with alpha-galactosidase. The absence of toxin interaction with isoglobotriaosylceramide (Gal alpha 1-3Gal beta 1-4Glc-Cer) isolated from canine intestine suggested that the GlcNAc residue present in the terminal Gal alpha 1-3Gal beta 1-4GLcNAc sequence common to all known toxin binding glycoconjugates is required for carbohydrate-specific recognition by Toxin A. These observations are consistent with the proposed carbohydrate binding specificity of Toxin A for the nonreducing terminal sequence, Gal alpha 1-3Gal beta 1-4GlcNAc.     

Isolation and comparative analysis of glycolipid fractions in bifidobacteria

A comparative TLC analysis of lipid extracts from Bifidobacterium longum B 379 M, B. bifidum 791, and B. adolescentis 94 BIM has been performed. It is demonstrated that carbohydrate-containing lipid components were present in the bacteria, which differed in their chromatographic mobility (Rf) from similar compounds isolated from actinomycetes Stomatococcus mucilaginosus PCM 2415T, Nocardiopsis dassonvillei PCM 2492, Propionibacterium propionicum PCM 2431, Saccharopolyspora hirsuta PCM 2279 (= ATCC 27875T), Rhodococcus equi PCMT 559 (= ATCC 3969), and Gordonia bronchialis PCM 2167. Polar lipids of bifidobacteria exhibited the closest similarity to their counterparts from propionic acid bacteria. Preparative chromatography (silica gel column I; elution with chloroform, acetone, and methanol) of the lipid extract of B. adolescentis 94 BIM made it possible to isolate fractions containing nonpolar lipids, glycolipids, and phospholipids. Further purification of the glycolipid fraction (column II; eluant, methanol gradient in chloroform) produced preparations of glycolipids and phospholipids. The preparations were studied by two-dimensional TLC using solvent systems chloroform-methanol-H2O MiLi Q (65 : 25 : 4, v/v/v) and n-butanol-acetic acid-H2O MiLi Q (60 : 20 : 20, v/v/v) for directions I and II, respectively. Two major glycolipids were revealed (G1 and G2), in addition to compounds characteristic of the polar lipid group and minor glycolipids (g), the latter being present in considerably lesser amounts.     

Lipid composition of prolamellar bodies and prothylakoids of wheat etioplasts

Prolamellar bodies and prothylakoids were fractionated from etioplasts of wheat ( Triticum aestivum L., cv. Starke II, Weibull) and characterized with emphasis on lipid composition. The two fractions contained the same lipid classes. Glycolipids (monogalactosyl diacylglycerol, digalactosyl diacylglycerol, and sulphoquinovosyl diacylglycerol) were the dominating complex lipids. Phospholipids (mainly phosphatidyl choline and phosphatidyl glycerol) constituted between 10 and 15 mol% of the total amounts of polar lipids. Free sterols and sterol esters were present in low amounts (ca 6 mol%). Saponins could not be detected. The contents of glycolipids and protochlorophyllide were higher in the prolamellar body fraction than in the prothylakoid fraction on a protein basis, as was the protochlorophyllide content on a glycolipid basis. The molar ratio of monogalactosyl diacylglycerol to digalactosyl diacylglycerol was higher in the prolamellar body fraction (1.8) than in the prothylakoid fraction (1.2).
Since the same chemical constituents were found in the two membrane fractions we propose that the difference in ultrastructure between prolamellar bodies and prothylakoids is due to different relative amounts of lipids (glycolipids), protochlorophyllide, and proteins in the two membrane systems.     

Consecutive analysis of sphingoglycolipids on the basis of sugar and ceramide moieties by high performance liquid chromatography

A quantitative consecutive method was developed for analysis of sphingoglycolipids in biological materials by high performance liquid chromatography (HPLC). Crude lipid extracts were separated into neutral and acidic fractions on a DEAE-Sephadex column. Glycolipid fractions were obtained by acetylation and Florisil column chromatography, and the acetylated glycolipids were N-p-nitrobenzoylated by treatment with p-nitrobenzoyl chloride in pyridine at 60 degrees C for 6 h. Excess reagent and by-products were removed by solvent partition and gel filtration. The glycolipid derivatives were analyzed by their absorption at 254 nm on Zorbax SIL, a silica gel column, with a gradient of 0.5--7% isopropanol in hexane-chloroform (2 : 1, v/v) at a flow rate of 0.5 ml/min. The detector response was linear with up to 60 nmol of injected glycolipids. The practical lower limit of detection was about 50 pmol. The derivatives were separated on the basis of their sugar chains. Effluents corresponding to each peak were collected and analyzed further on the basis of their lipid portion on mu-Bondapak C18, a reversed phase column. This combined procedure was applied to the analysis of erythrocyte glycolipids. Samples containing as little as 20 micrograms of glycolipids could be analyzed by this method.     

The fatty acid and monosaccharide compositions of three neutral and three phosphorylated glycolipids isolated from Leishmania donovani promastigotes grown in a chemically defined medium

Several lipids and macromolecular lipoconjugates of Leishmania spp. have now been well characterized; however, the glycolipids of L. donovani have not been thoroughly examined. In the present study, 3 neutral and 3 phosphorylated glycolipids were detected in promastigote forms of the organism grown in a chemically defined medium. The fatty acid and sugar compositions of these glycolipids, isolated and purified by adsorption column chromatography and thin-layer chromatographic procedures, were identified and quantified by gas-liquid chromatography and mass spectrometry. Myristate (14:0), palmitate (16:0), palmitoleate (16:1), stearate (18:0), oleate (18:1), and linoleate (18:2) were the major fatty acids in all 6 glycolipids. Arabinose, mannose, glucose, and galactose were detected in the glycolipids. The biochemical nature of these lipids suggested that the major components in the isolated preparations of the 6 glycolipids are diacylglycerophospholipids, distinct from the major precursors of macromolecular lipoconjugates such as the lipid anchors of cell surface antigens that have been reported. These appear to be terminal products of lipid biosynthesis in this parasite.     

Lipid Composition of Growing and Starving Cells of Arthrobacter crystallopoietes

The lipid composition of growing and starving cells of Arthrobacter crystallopoietes was compared. Although the lipid composition of the two cell types was similar, the amount of total lipids recovered from the starving cells was 30.4% less than that recovered from the growing cells. The loss of lipids, as compared to the loss of total cell mass during starvation, was (i) proportional to the loss of the cell mass (phosphatidylinositol, phosphatidylglycerol-2, and cardiolipin), (ii) greater than the loss in cell mass (neutral lipids, "glycophospholipids," and phosphatidic acid), or (iii) less than the loss in cell mass (coenzyme Q, glycolipids, and phosphatidylglycerol-1).     

Composition of lipids in roots, stalks, leaves and flowers of Eichhornia crassipes (Mart.) Solms

The lipid contents of the roots, leaf stalks, leaves and flowers of Eichhornia crassipes (Mart.) Solms (water-hyacinth) were 1.6, 0.9, 14.9 and 5.7%, respectively, on a dry-weight basis. Non-polar lipids were half the total, while glycolipids and phospholipids in approximately equal proportions constituted the remainder, except in leaf stalks, where glycolipids were a larger fraction. Among the non-polar lipids, triacyglycerols predominated, except for pigments in the leaves. Monogalactosyldiglycerides and digalactosyldiglycerides were the major glycolipids. The main phospholipids were phosphatidylcholine in the roots, phosphatidylglycerol in the leaf stalks and leaves, and phosphatidylethanolamine in the flowers. The major fatty acids were palmitic and linoleic in the roots, linoleic in the leaf stalks, palmitic in the leaves, and linolenic and linoleic in the flowers.     

Glycolipid-lectin interactions: reactivity of lectins from Helix pomatia, Wisteria floribunda, and Dolichos biflorus with glycolipids containing N-acetylgalactosamine

The autoradiographic detection of 125I-labeled lectins binding to glycolipids on thin-layer chromatograms can be used to rapidly analyze total glycolipid extracts of cells or tissues for specific oligosaccharide structures. The Helix pomatia lectin which binds with high affinity to terminal alpha-linked GalNAc residues did not bind to globoside (terminal beta 1-3GalNAc) but did bind the ganglioside GM2 and its asialo derivative which have terminal beta 1-4GalNAc residues. The lectin from Dolichos biflorus bound specifically to the Forssman glycolipid with relatively low affinity. The lectin from Wisteria floribunda was bound to Forssman glycolipid, globoside, and the asialo derivative of the ganglioside GM2. The interactions of these lectins with the glycolipid-derived, 3H-labeled oligosaccharides was also analyzed by affinity chromatography. The results indicated that the reactivity of multivalent carbohydrate-binding proteins with polyvalent surfaces of glycolipids is strong enough to permit detection of low-affinity interactions that may not be observed in binding assays that are based on carbohydrate-protein interactions in solution. The autoradiographic analysis of 125I-Helix pomatia lectin binding to thin-layer chromatograms of total lipid extracts from human erythrocyte membranes detected the quantitative differences in the A-active glycolipids from type A1 and A2 cells.