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THE ISOLATION AND PARTIAL CHARACTERIZATION OF THE GLYCOLIPIDS OF BP8/C3H ASCITES-SARCOMA CELLS.

ABSTRACT: 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.

SUBMITTER: GRAY GM

PROVIDER: S-EPMC1206411 | biostudies-other | 1965 Jan

REPOSITORIES: biostudies-other

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Project description:Sialomucins are the dominant components of the cell surfaces of some carcinoma ascites cells and have been postulated to inhibit recognition of tumours by the immune system. The sialomucin ASGP-1 (ascites sialoglycoprotein-1) of the 13762 rat mammary adenocarcinoma is associated with the cell surface as a complex with a concanavalin-A-binding glycoprotein called ASGP-2. This sialomucin complex has been purified from ascites cell microvilli by extraction with Triton X-100 and CsCl density-gradient centrifugation. ASGP-1 (which has been purified previously) and ASGP-2 were dissociated in 6 M-guanidine hydrochloride and separated by gel filtration. The molecular mass of the undenatured detergent complex of ASGP-2, estimated by gel filtration and velocity sedimentation in Triton X-100, was 148 kDa. Since the apparent molecular mass by SDS/polyacrylamide-gel electrophoresis was about 120 kDa, ASGP-2 must be a monomer as extracted from the membrane. Studies of its chemical composition indicate that it contains about 45% carbohydrate by weight, including both mannose and galactosamine. Alkaline borohydride treatment of ASGP-2 converted approx. half of the N-acetylgalactosamine to N-acetylgalactosaminitol, demonstrating the presence of O-linked oligosaccharides. Analyses of mannose-labelled Pronase glycopeptides from ASGP-2 by lectin-affinity chromatography on concanavalin A and leucocyte-agglutinating phytohaemagglutinin suggested that 40% of the label was present in high-mannose/hybrid oligosaccharides, 20% in triantennary oligosaccharides substituted on the C-2 and C-4 mannose positions and 40% in tri- or tetra-antennary oligosaccharides substituted on C-2 and C-6. The presence of polylactosamine sequences on these oligosaccharides was suggested by lectin blots and by precipitation from detergent extracts with tomato lectin. From chemical analyses and lectin-affinity studies, we estimate that ASGP-2 contains four high-mannose and 13 complex N-glycosylated oligosaccharides, plus small amounts of polylactosamine and O-linked oligosaccharides. The presence of four different classes of oligosaccharides on this glycoprotein suggests that it will be an interesting model system for biosynthetic comparisons of the different glycosylation pathways.

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THE ISOLATION AND PARTIAL CHARACTERIZATION OF THE GLYCOLIPIDS OF BP8/C3H ASCITES-SARCOMA CELLS.

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