Separation of neutral glycosphingolipids and sulfatides by thin-layer chromatography

Skipski, Vladimir P.; Smolowe, Anson F.; Barclay, Marion

doi:10.1016/s0022-2275(20)39557-2

Cited by 66 publications

(3 citation statements)

References 26 publications

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“…Each 2 μL aliquot of 2 mg/mL reaction mixture was loaded onto a 20 cm × 20 cm plastic TLC plate precoated with 0.2 mm silica gel (Macherey-Nagel, Düren, Germany) and developed with a solvent system consisting of n -butanol:formic acid:water (4:7:1, v/v/v) at 25 °C. The developed plate was stained by spraying with orcinol–sulfuric acid reagent (0.2 g of orcinol, 20 mL of 98% H 2 SO 4 , 100 mL of methanol) and heated at 150 °C until the bands were obviously visible …”

Section: Methodsmentioning

confidence: 99%

“…The developed plate was stained by spraying with orcinol−sulfuric acid reagent (0.2 g of orcinol, 20 mL of 98% H 2 SO 4 , 100 mL of methanol) and heated at 150 °C until the bands were obviously visible. 36 3-Amino-9-ethylcarbazole (AEC) Labeling and HPLC Analysis of κ-Carrageenan Hydrolysate. Five milligrams of κ-carrageenan oligosaccharides obtained at different intervals was dissolved in 500 μL of deionized water.…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

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Electrospray Ionization Mass Spectrometric Analysis of κ-Carrageenan Oligosaccharides Obtained by Degradation with κ-Carrageenase from Pedobacter hainanensis

Sun

Liu

Jiang

et al. 2014

J. Agric. Food Chem.

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κ-Carrageenan was degraded with a novel κ-carrageenase isolated from Pedobacter hainanensis, which was first isolated from seaside soil under the stacks of red algae in Hainan province of China. The κ-carrageenase was detected with a molecular weight of ∼55 kDa estimated from SDS-PAGE and yielded enzymatic activity of 700.53 units/mg of protein under the conditions of pH 7.0 and 40 °C. Analysis of the degradation products by TLC and HPLC indicated that the enzyme degraded κ-carrageenan to sulfated oligosaccharides with even-numbered degree of polymerization, of which the tetrasaccharide was the major product. All the degradation components during different time courses were analyzed by ESI-MS, and their structures were assigned. Structural analysis by CID MS/MS revealed that each carrageenan oligosaccharide was composed of An-G4S-type neocarrabiose units, which consisted of a 3,6-anhydro-α-d-galactose (An) residue in the nonreducing end and a β-d-galactose-4-sulfate (G4S) residue in the reducing end. These results demonstrated that the κ-carrageenase cleaved κ-carrageenan at the internal β-1,4 linkage of κ-carrageenan. This enzymatic degradation offers an alternative approach to prepare κ-carrageenan oligosaccharides, which could be used as a powerful tool for further study on biological activity-structure relationship and thorough industrial exploitation of κ-carrageenan.

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Section: Methodsmentioning

confidence: 99%

Section: ■ Materials and Methodsmentioning

confidence: 99%

Electrospray Ionization Mass Spectrometric Analysis of κ-Carrageenan Oligosaccharides Obtained by Degradation with κ-Carrageenase from Pedobacter hainanensis

Sun

Liu

Jiang

et al. 2014

J. Agric. Food Chem.

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“…The lipids were resolved on silica gel H plates developed in chloroform/ methanol/acetic acid/water (25:15:4:2). The plates were developed in the same solvent system three times (50). Spots were identified by comparison to the migration patterns of known standards following staining with Dragendorf, ninhydrin, and sulfuric acid charring.…”

Section: Release Of Chicken Epiphyseal Growth Plate Chondrocytes and MVmentioning

confidence: 99%

Chondrocytes Utilize a Cholesterol-Dependent Lipid Translocator To Externalize Phosphatidylserine

et al. 2006

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During endochondral ossification, growth plate chondrocytes release plasma membrane (PM) derived matrix vesicles (MV), which are the site of initial hydroxyapatite crystal formation. MV constituents which facilitate the mineralization process include the integral membrane ectoenzymes alkaline phosphatase (ALPase) and nucleotide pyrophosphatase phosphodiesterase (NPP1/PC-1), along with a phosphatidylserine-(PS-) rich membrane surface that binds annexins and calcium, resulting in enhanced calcium entry into MV. In this study, we determined that chick growth plate MV were highly enriched in membrane raft microdomains containing high levels of cholesterol, glycophosphatidylinositol-(GPI-) anchored ALPase, and phosphatidylserine (PS) localized to the external leaflet of the bilayer. To determine how such membrane microdomains arise during chondrocyte maturation, we explored the role of PM cholesterol-dependent lipid assemblies in regulating the activities of lipid translocators involved in the externalization of PS. We first isolated and determined the composition of detergent-resistant membranes (DRMs) from chondrocyte PM. DRMs isolated from chondrocyte PM were enhanced in ganglioside 1 (GM1) and cholesterol as well as GPI-anchored ALPase. Furthermore, these membrane domains were enriched in PS (localized to the external leaflet of the bilayer) and had significantly higher ALPase activity than non-cholesterol-enriched domains. To understand the role of cholesterol-dependent lipid assemblies in the externalization of PS, we measured the activities of two lipid transporters involved in PS externalization, aminophospholipid translocase (APLT) and phospholipid scramblase (PLSCR1), during maturation of a murine chondrocytic cell line, N1511. In this report, we provide the first evidence that maturing chondrocytes express PLSCR1 and have scramblase activity. We propose that redistribution of PS is dependent on an increase in phospholipid scramblase activity and a decrease in APLT activity. Lastly, we show that translocator activity is most likely to be modulated by membrane cholesterol levels through a membrane raft microdomain. † This work was supported by USPHS Grant DE13576. * Address correspondence to this author. . battagli@biochem.dental.upenn.edu. HHS Public Access Author manuscriptBiochemistry. Author manuscript; available in PMC 2016 January 29. Author Manuscript Author ManuscriptAuthor Manuscript Author ManuscriptVertebrate skeletal growth occurs largely through a highly regulated process known as endochondral ossification. During this process mesenchymal cells condense and differentiate into chondrocytes to form a cartilage template. Chondrocytes proliferate and then undergo a complex maturation process associated with an increase in cell size (hypertrophy). This hypertrophic stage is characterized by upregulation of the expression of genes encoding ALPase, 1 type X collagen, and matrix metalloproteinase-13, followed by mineralization of the extracellular matrix. The zones of proliferating, prehypertro...

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Phosphatidyl Inositol and Complex Inositides in Autolysed Baker's Yeast

Trevelyan

1968

Journal of the Institute of Brewing

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Complex glycosphingolipids present in extracts of toluene-autolysed baker's yeast were removed by solvent fractionation with a I : I mixture of chloroform and methanol. Phospholipids which were soluble in chloroform-methanol 1:1, but insoluble in isopropanol, were converted into the sodium salts, and crystallization of these from ether-methanol gave phosphatidyl Inositol, free from cardiolipin.Further purification was effected by adsorbing the phospholipid on silicic acid, and developing the initially dry column with chloroform-methanol-water. The glycosphingolipid fraction was resolved into residual phosphatidyl inositol and two glycolipid components, by chromatography on Florisil. These complex inositides possibly derive from the yeast cell wall, and, if so, they may influence the hydrophllic/ hydrophobic character of the cell surface if located on the outer side or, if on the inner side, may constitute a link between the cell membrane and the cell wall.

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Separation of neutral glycosphingolipids and sulfatides by thin-layer chromatography

Cited by 66 publications

References 26 publications

Electrospray Ionization Mass Spectrometric Analysis of κ-Carrageenan Oligosaccharides Obtained by Degradation with κ-Carrageenase from Pedobacter hainanensis

Electrospray Ionization Mass Spectrometric Analysis of κ-Carrageenan Oligosaccharides Obtained by Degradation with κ-Carrageenase from Pedobacter hainanensis

Chondrocytes Utilize a Cholesterol-Dependent Lipid Translocator To Externalize Phosphatidylserine

Phosphatidyl Inositol and Complex Inositides in Autolysed Baker's Yeast

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