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1.
Five major neutral glycolipids, GL-1-GL-5, were isolated from the the mouse small intestine. Their structures and distribution were determined by permethylation analysis, sequential degradation with exoglycosidases and/or immunohistochemistry. The molar ratio of GL-1, GL-2, GL-3, GL-4 and Gl-5 in the whole small intestine was 1:0.04:0.03:0.42:0.02. The structures of GL-1 and GL-4 present in epithelial cells were reported previously to be glucosyl ceramide and asialo GM1, respectively (Umesaki, Y., Suzuki, A., Kasama, T., Tohyama, K., Mutai, M. and Yamakawa, T. (1981) J. Biochem. 90, 1731–1738). GL-5, also present in the epithelial cells, was fucosyl asialo GM1, and fucose was shown to be linked to terminal galactose of asialo GM1 in the manner of α(1–2) bond. GL-2 and GL-3, present in the residual tissue after scraping the mucosa, were determined to be globoside and Forssman glycolipid, respectively. Both globoside and Forssman glycolipid of the non-epithelial tissue had non-hydroxy fatty acid (C16–C24) in combination with sphingosine (C18) as the ceramide components, in contrast with the ceramide structures of the epithelial glycolipids, which contained α-hydroxy fatty acids in combination with phytosphingosine. Immunohistochemical staining using anti-glycolipid antibodies confirmed the distribution of asialo GM1 and fucosyl asialo GM1, and Forssman glycolipid in the epithelial and non-epithelial tissue, respectively.  相似文献   

2.
A panel of five plant lectins with different binding specificities was used to determine if plant lectins could bind specifically to membrane-associated glycolipids. Ricinis communis and wheat germ agglutinins both bound specifically to mixed brain gangliosides and globoside I from human erythrocytes. Wheat germ agglutinin also bound to ganglioside GM1 and human erythrocyte ceramide trihexoside, but not to ceramide dihexoside, mono-, or digalactosyl diglycerides. Concanavalin A bound to liposomes with or without glycolipid substituents, and this binding was partially inhibited by α-methyl mannoside. This study indicates that lectins can specifically recognize and bind to certain glycolipids in membranes.  相似文献   

3.
The exposure of glycolipids in Pk and p red cells was studied by the galactose oxidase/ NaB2H4 and galactose oxidase/NaB3H4 surface labeling techniques. The major glycolipid in Pk cells, ceramide trihexoside was efficiently labeled when high amounts of galactose oxidase were used. In contrast, the major glycolipid in p cells, ceramide dihexoside was not oxidized by galactose oxidase. However, minor components with longer oligosaccharide chains were readily labeled in p cells by the galactose oxidase/NaB3H4 method.Abbreviations CDH ceramide dihexoside, LacCer - CTH ceramide trihexoside, GbOse3Cer  相似文献   

4.
A radioimmunoassay that measures Leb-active glycolipids in human plasma has been developed using antiserum from a goat immunized with a Leb blood group hapten, lacto-N-difucohexaose I, conjugated to polylysine. Binding by the antiserum of lacto-N-difucohexaose I conjugated to 125I-labeled bovine serum albumin is specifically inhibited by Leb-active ceramide hexasaccharide. Plasma levels of the glycolipid are quantitated by comparing the inhibitory activity of plasma with that of the purified Leb-active glycolipid. Plasma samples from 35 blood group O Le(a ? b +) individuals contain Leb-active ceramide hexasaccharide at an average concentration of 0.9 μg/ml (range: 0.2 to 2.5 μg/ml); no Leb-active glycolipid (less than 0.02 μg/ml) could be detected in plasma from blood group O Le(a + b?) or O Le(a? b?) individuals. Plasma from A1 Le(a ? b+) individuals contains less Leb-active glycolipid than plasma from A2 Le(a? b+) individuals: its level in 19 samples of A, Le(a? b+) plasma averages 0.2 μg/ml (range: 0.1 to 0.45 μg/ml), and its level in 9 samples of A2 Le(a? b+) plasma averages 1.1 μg/ml (range 0.8 to 1.3 μg/ml). About one-third of the total Leb-active glycolipid in whole blood is associated with erythrocytes and the rest is found in plasma.  相似文献   

5.
Oxidation of glycolipids in liposomes by galactose oxidase   总被引:1,自引:0,他引:1  
Small unilamellar phosphatidylcholine vesicles containing globo-series glycolipids were labeled by the galactose oxidase/NaB[3H]4 procedure. The major glycolipid of human red cells, globoside, was the best substrate for galactose oxidase both in vesicles and in tetrahydrofuran-containing buffer. The oxidation rates of membrane-bound ceramide trihexoside and Forssman glycolipid were one-fourth and one-tenth, respectively, of the oxidation rate of globoside. Membrane-bound ceramide dihexoside was not a substrate for galactose oxidase, although it was readily oxidized in tetrahydrofuran-containing buffer. Soluble sialoglycoproteins and membrane-incorporated glycophorin A stimulated the oxidation of globoside-containing vesicles, whereas membrane-bound GD1a ganglioside had no effect on globoside oxidation.  相似文献   

6.
Four glycolipids have been isolated from three fractions of pig blood. The glycolipids were presumably cerebroside, diglycosyl ceramide, triglycosyl ceramide, and globoside. The blood fractions were erythrocytes and plasma high and low density lipoproteins. Fatty acid distributions were determined for each glycolipid as a means to assist in identifying relationships among the several glycolipids. Normal fatty acids predominated in all glycolipids except the globosides from erythrocytes in which the amount of hydroxy acids was slightly greater than the amount of normal acids. Hydroxy acids appeared to be present in all the glycolipids, but the concentration was very low in cerebrosides isolated from high density lipoproteins and erythrocytes, and in diglycosyl ceramide and globoside of the low density lipoproteins. In general, the average fatty acid chain length increased from cerebroside to globoside. This was most apparent in erythrocytes and also greater for normal acids than for hydroxy acids. Fatty acid distributions of erythrocyte glycolipids had sufficient variation to make a metabolic relationship by simple addition of a hexose appear doubtful. While the fatty acid distributions found in plasma lipoproteins were more similar, some means of acyl group selection is probably present for either the synthesis or degradation of these glycolipids.  相似文献   

7.
Yeast membranes incorporate radioactivity from GDP[14C]mannose into various glycolipids. These can be separated by thin layer chromatography into at least seven components.The major component has been identified previously as dolichyl monophosphate mannose. Only one additional component is not sensitive to mild alkaline saponification, but is hydrolyzed instead under mild acidic conditios. This latter glycolipid has all the characteristics of a polyprenyl diphosphate oligosaccharide with a sugar moiety of more than 12 hexose units. It runs like dolichyl diphosphate derivatives on a DEAE column and evidence is presented that the lipid moiety is a polyprenol.When radioactive Dol-PP-di-N-acetylchitobiose is incubated with yeast membranes in the presence of non-radioactive GDPmannose a small amount of a larger lipid oligosaccharide is formed besides the previously-described Dol-PP-(GlcNAc2 mannose. This oligosaccharide has all the properties of the glycolipid described above. Its formation is greatly increased when Triton is omitted from the incubation. Radioactivity of the polyprenyl diphosphate [14C]oligosaccharide is transferred to ethanol-insoluble material, most likely endogenous membrane glycoproteins.  相似文献   

8.
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 (R f ) 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.  相似文献   

9.
Monosialogangliosde liposomes are rapidly taken up by the liver as compared to dicetylphosphate, phosphatidic acid or neutral liposomes. Asialoganglioside GM 1 liposomes are taken up with the same avidity as ganglioside GM 1 liposomes. Competition experiments with asialofetuin suggest that this uptake is mediated by specific recognition of the terminal galactose residues of the glycolipid liposomes by the receptor present on the plasma membrane of the parenchymal cells of liver. Thus liposomes containing glycolipids with terminal β-galactosyl residues should provide an approach for specifically directing biologically active molecules to liver parenchymal cells.  相似文献   

10.
We analysed the glycolipid composition of glioma cells (N-370 FG cells), which are derived from a culture of transformed human fetal glial cells. The neutral and acidic glycolipid fractions were isolated by column chromatography on DEAE-Sephadex and analysed by high-performance thin-layer chromatography (HPTLC). The neutral glycolipid fraction contained 1.6 µg of lipid-bound glucose/galactose per mg protein and consisted of GlcCer (11.4% of total neutral glycolipids), GalCer (21.5%), LacCer (21.4%), Gb4 (21.1%), and three unknown neutral glycolipids (23%). These unknown glycolipids were characterized as Lewisx (fucosylneolactonorpentaosyl ceramide; Lex), difucosylneolactonorhexaosyl ceramide (dimeric Lex), and neolactonorhexaosyl ceramide (nLc6) by an HPTLC-overlay method for glycolipids using specific mouse anti-glycolipid antibodies against glycolipid and/or liquid-secondary ion (LSI) mass spectrometry. The ganglioside fraction contained 0.6 µg of lipid-bound sialic acid per mg protein with GD1a as the predominant ganglioside species (83% of the total gangliosides) and GM3, GM2, and GM1 as minor components. Trace amounts of sialyl-Lex and the complex type of sialyl-Lex derivatives were also present. Immunocytochemical studies revealed that GD1a and GalCer were primarily localized on the surface of cell bodies. Interestingly, Lex glycolipids and sialyl-Lex were localized not only on the cell bodies but also on short cell processes. Especially, sialyl-Lex glycolipid was located on the tip of fine cellular processes. The unique localization of the Lex glycolipids suggests that they may be involved in cellular differentiation and initiation of cellular growth in this cell line.  相似文献   

11.
Polarity of the Forssman glycolipid in MDCK epithelial cells   总被引:3,自引:0,他引:3  
To determine whether epithelial plasma membrane glycolipids are polarized in a manner analogous to membrane proteins, MDCK cells grown on permeable filters were analyzed for the expression of Forssman ceramide pentasaccharide, the major neutral glycolipid in these cells. In contrast to a recent report which described exclusive apical localization of the Forssman glycolipid (Hansson, G.C., Simons, K. and Van Meer, G. (1986) EMBO J. 5, 483-489), immunofluorescence and immunoelectron microscopic staining revealed the Forssman glycolipid on both the apical and basolateral surfaces of polarized cells. Immunoblots indicated that the Forssman antigen was detectable only on glycolipids and not on proteins. Analysis of metabolically labeled glycolipids released into the apical and basal culture medium, either as shed membrane vesicles or in budding viruses, also demonstrated the presence of the Forssman glycolipid on both apical and basolateral membranes of polarized cells. Quantitation of the released glycolipid indicated that the Forssman glycolipid was concentrated in the apical membrane. These results are consistent with previous reports which described quantitative enrichment of glycolipids in the apical domain of several epithelia.  相似文献   

12.
Gangliosides in the external surface of intact synaptosomes from rat brain cortex have been studied by oxidation of exposed galactose and galactosamine groups with galactose oxidase followed by reduction with labeled sodium borohydride. Purified synaptosomes were labeled, disrupted by osmotic shock, and the particulate components fractionated on diatrizoate to give four synaptosomal membrane fractions (A-D) and a mitochondrial pellet (E). Fractions A and B represent synaptosomal plasma membranes. When intact synaptosomes were labeled, the major portion of the total radioactivity incorporated into ganglioside fraction was found to be in G M1 3 species. With isolated membrane fractions little selectivity was seen: (1) more label was present compared to intact synaptosomes, and (2) zones corresponding to GM2, GM1, GD1a, GD1b were the major gangliosides labeled. The results confirm the conclusion that membrane fractions A and B are derived from the exposed synaptosome surface and also show that GM1 is the major ganglioside species available for enzyme oxidation at the surface.  相似文献   

13.
Extracts of the human intestinal tumor cell line SW1116 were able to stimulate the incorporation of (14C) fucose from GDP-(14C) fucose into organically soluble glycolipid. The reaction required a purified glycolipid preparation from human meconium as lipid acceptor. The active glycolipid co-migrated with standard globoside on high performance thin-layer chromatography (HPTLC) and had molecular species (M + H) under fast-atom bombardment mass spectrometry of 1199, 1245 and 1269. Globoside itself was inactive and asialo GM1b had low activity. The radioactive products co-purified with Lewis a and Lewis b and co-migrated principally (60-90%) with Lewis b monoclonal antibody binding cellular glycolipids on HPTLC. Analysis of fucosidase digests suggested the presence of two different fucosyl-hexose linkages one of which was susceptible to cleavage. We conclude that the data are consistent with fucosylation of lactotetraosyl ceramide to Lewis a and Lewis b antigenic glycolipids.  相似文献   

14.
The binding of uropathogenic Escherichia coli to the globo series of glycolipids via P pili is a critical step in the infectious process that is mediated by a human-specific PapG adhesin. Three classes of PapG adhesins exist with different binding specificities to Galα4Gal-containing glycolipids. The structural basis for PapG recognition of the human glycolipid receptor globoside was investigated by using soluble saccharide analogues as inhibitors of bacterial haemagglutination. The minimum binding epitope was confirmed as the Galα4Gal moiety, but parts of the GalNAcβ and glucose residues, which flank the Galα4Gal in globoside (GbO4), were also shown to be important for strong binding. Furthermore, the same five hydroxyl groups of Galα4Gal in globotriasyl ceramide that were recognized by a previously characterized PapG variant were also recognized by the human-specific PapG in binding the GbO4 that dominates In the human kidney. Saccharide analogues that blocked haemagglutination also blocked the adherence of human uropathogenic E. coli to human kidney sections. Knowledge of the molecular details of the PapG-GbO4 interaction will make it possible to design antiadherence therapeutics.  相似文献   

15.
Incubating white matter membranes with UDP-N-acetyl-[14C]glucosamine in the presence of Mg2+ and AMP resulted in the labeling of two major glycolipids, a minor glycolipid and several membrane-associated glycoproteins. The addition of AMP protected the labeled sugar nucleotide from degradation by a membrane-bound sugar nucleotide pyrophosphatase activity. While no labeled oligosaccharide lipid was recovered in a CHCl3CH3OHH2O (10:10:3) extract after incubating with only UDP-N-acetyl-[14C] glucosamine, Mg2+, and AMP, the inclusion of unlabeled GDP-mannose led to the formation of an N-acetyl-[14C]glucosamine-labeled oligosaccharide lipid that was soluble in CHCl3CH3OHH2O (10:10:3). The [GlcNAc-14C]oligosaccharide unit was released by treatment with 0.1 N HCl in 80% tetrahydrofuran at 50 °C for 30 min and appears to have the same molecular size as the lipid-linked [mannose-14C] oligosaccharide, formed enzymatically by white matter membranes as judged by their elution behavior on Bio-Gel P-6. The incorporation of N-acetyl-[14C]glucosamine into glycolipid was stimulated by exogenous dolichol monophosphate, but inhibited by UMP or tunicamycin, a glucosamine-containing antibiotic. Although UMP and tunicamycin drastically inhibited the labeling of glycolipid, these compounds had very little effect on the labeling of glycoproteins. The major glycolipids have the chemical and Chromatographic characteristics of N-acetylglucosaminylpyrophosphoryldolichol and N,N′-diacetylchitobiosylpyrophosphoryldolichol. When the labeled glycoproteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, four labeled polypeptides were observed, having apparent molecular weights of 145,000, 105,000, 54,000, and 35,000. Virtually all of the N-acetyl-[14C]glucosamine was released when the labeled glycopeptides, produced by pronase digestion, were incubated with an exo-β-N-acetylglucosaminidase, indicating that all of the N-acetyl-[14C]glucosamine incorporated under these conditions is attached to white matter membrane glycoproteins at nonreducing termini.  相似文献   

16.
From 8 1 of human plasma of blood-group A Lea nonsecretors three different Lea blood-group active ceramide pentasaccharides (a total of 4.65 mg) have been isolated, all revealing glucose, galactose, N-acetylglucosamine and fucose in molar ratios of 1 : 2 : 1 : 1 as determined by gas liquid chromatography. A fourth blood-group active fraction (0.72 mg) represents a mixture of a Lea active ceramide pentasaccharide and an A active ceramide hexasaccharide (molar ratio 7.7 : 2.3 as calculated from the content of different aminosugars). Additionally, two different globosides, two different hematosides and a new N-acetylglucosamine containing ceramide tetrasaccharide were obtained. All 9 glycolipid fractions demonstrated homogeneity in analytical high performance thin layer chromatography (HPTLC) using 4 different solvent systems. 0.2 μg of each Lea active glycolipid completely inhibited the agglutination of O Le(a + b ?) erythrocytes by 50 μl of 4 hemagglutinating units of caprine anti Lea serum. At least 0.04 μg of each Lea antigen are sufficient for incubation to convert 9 × 107 O Le(a?b?) erythrocytes into Lea-positive cells. Mainly due to the relatively low content of the blood-group A glycolipid in plasma (0.17 mg/8 1), previously negative erythrocytes readily become agglutinable by anti Lea sera and not by anti A sera after incubation with appropriate plasma.  相似文献   

17.
Cultured adrenergic neurons from newborn rat superior cervical ganglia bind the lectin soybean agglutinin (SBA) at a fivefold higher density than the same neurons which have been induced to become cholinergic (M. Schwab and S. L. Landis, 1981, Dev. Biol.84, 67–78). In the present experiments, the binding sites for this lectin on the surfaces of living neurons were identified by labeling the surfaces with the galactose oxidase-[3H]sodium borohydride reduction technique, with and without prior incubation with the lectin. SBA binds to and inhibits the labeling of two neutral glycolipids, a glycolipid comigrating with globoside on thin-layer chromatograms and an unidentified glycolipid. When neuronal proteins are extracted and separated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate, SBA shows only very faint labeling of this fraction. Thus the SBA binding sites on these neurons appear to be two neutral glycolipids. Further support for this conclusion comes from the finding that the two neutral glycolipids detected by SBA are present in smaller amounts or are less accessible on the cholinergic than on the adrenergic neurons as measured by surface labeling. In addition to the difference in neutral glycolipids, external labeling revealed quantitative differences in the major gangliosides of the two types of cultured neurons. Thus, by using pure cultures of sympathetic neurons which can be induced to become either adrenergic or cholinergic, specific glycolipid profiles were correlated with the two neurotransmitter phenotypes.  相似文献   

18.
Glycolipid composition of purified plasma membranes from rat ascites hepatomas, two island-forming cell-lines and two cell-lines of the free-type, and normal rat liver were compared. Ceramide monohexoside (CMH), ceramide dihexoside (CDH), and hematoside (GM3) were found in normal rat liver cell membranes. The island-type hepatomas contained ceramide trihexoside (CTh) and globoside besides CMH, CDH, and GM3. The free-type of hepatomas were characterized by the presence of asialo-type gangliosides but not GM3. The free-type of hepatomas were characterized by the presence of asialo-type gangliosides but not GM3. Blood group H active fucolipid was a major glycolipid in the free-type of ascites hepatoma cell (AH 7974 F). The increase of glycolipid content in cell membranes seemed to be accompanied with a decrease of cell adhesiveness.  相似文献   

19.
Monoclonal B cell hybridoma cell lines express glycolipids characteristic of both the myeloma and normal lymphocyte parents. The neutral glycolipids from hybridoma cell lines metabolically radiolabeled with [14C]galactose plus [14C]glucosamine separate by high performance thin layer chromatography into patterns that may reflect differences in glycolipid expression among B cell subsets. Gas chromatography/mass spectrometry of oligosaccharides released by trifluoroacetolysis from neutral glycolipids extracted from 10(9) clonally expanded hybridoma cells reveals the carbohydrate composition of the major glycolipid components detected by thin layer chromatography. Glycolipids differentially expressed among six cell lines analyzed include monohexosylceramide, lactosylceramide, globotriaosylceramide, globoside, asialo-GM2, and 2'fucosyllactosylceramide. The latter compound has not been described previously in cells from the mouse.  相似文献   

20.
The two clonal murine muscle cell lines G7 and G8, originally derived from the M114 line [20], represent unique models for comparative studies of myogenesis. Glycolipid synthesis was examined during differentiation using [3H]-galactose and [3H]-glucosamine as precursors. Upon G7 contact glucosylceramide labeling increased and nLcOse5Cer labeling stopped. During membrane fusion, glucosylceramide labeling stopped and lactosylceramide became the major synthetic product. G8 cells presented a different pattern, with increased labeling of GbOse3Cer during myogenesis. The major ganglioside synthesized by both myoblasts was GM3, and more complex structures were observed following completion of myotube formation. Total glycopeptide labeling increased when G8 myoblasts fused and remained elevated in myotubes, whereas no differences during fusion of G7 cells were noted. Upon comparison of the two clonal lines, the only consistent observation was a significant increase in the synthesis of total gangliosides and neutral glycolipid during cell contact and membrane fusion (p < 0.02). The results suggest that changes in the synthesis of specific glycolipid structures during myogenesis are unique to each muscle cell line examined. However, transient increases in synthesis of total myoblast gangliosides and neutral glycolipids may be a more general phenomenon, possibly by curbing proliferation or by altering myoblast membrane fluidity characteristics during differentiation.Abbreviations MG6 VI3NeuAc-V4Gal-IV3GlcNAc-nLcOse4Cer - TLC thin-layer chromatography - HPTLC high performance thin-layer chromatography - Gal galactose - GlcNH glucosamine - PBS phosphate buffered saline - CK creatine kinase  相似文献   

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