TAILIEUCHUNG - Báo cáo khoa học: Lipids and glycosphingolipids in caveolae and surrounding plasma membrane of primary rat adipocytes

We havemade a comprehensive and quantitative analysis of the lipid composition of caveolae from primary rat fat cells and compared the composition of plasma membrane inside and outside caveolae. We isolated caveolae from purified plasmamembranesusingultrasonication incarbonatebuffer to disrupt the membrane, or extraction with nonionic detergent, followed by density gradient ultracentrifugation. The carbonate-isolated caveolae fraction was further immunopurified using caveolin antibodies. | Eur. J. Biochem. 271 2028-2036 2004 FEBS 2004 doi Lipids and glycosphingolipids in caveolae and surrounding plasma membrane of primary rat adipocytes Unn Ortearen1. Maraareta Karlsson1 Natascha Blazic2 Maria Blomavist2 Fredrik H. Nvstrom1 BBB Johanna Gustavsson1 Pam Fredman2 and Peter Strẵlíors1 1 Department of Cell Biology and Diabetes Research Centre Linkoping University Sweden department of Clinical Neuroscience Sahlgrenska University Hospital Molndal Goteborg University Molndal Sweden We have made a comprehensive and quantitative analysis of the lipid composition of caveolae from primary rat fat cells and compared the composition of plasma membrane inside and outside caveolae. We isolated caveolae from purified plasma membranes using ultrasonication in carbonate buffer to disrupt the membrane or extraction with nonionic detergent followed by density gradient ultracentrifugation. The carbonate-isolated caveolae fraction was further immunopurified using caveolin antibodies. Carbonate-isolated caveolae were enriched in cholesterol and sphingomyelin and the concentration was three-and twofold higher respectively in caveolae compared to the surrounding plasma membrane. The concentration of glycerophospholi-pids was similar suggesting that glycerophospholipids constitute a constant core throughout the plasma membrane. The composition of detergent-insoluble fractions of the plasma membrane was very variable between preparations but strongly enriched in sphingomyelin and depleted of glycerophospholipids compared to carbonate-isolated caveolae indicating that detergent extraction is not a suitable technique for caveolae preparation. An average adipocyte caveola contained about 22 X 103 molecules of cholesterol X 103 of sphingomyelin and 23 X 103 of glycerophospholipid. The glycosphingolipid GD3 was highly enriched in caveolae whereas GM3 GM1 and GD1a were present inside as well as outside the caveolae membrane. GD1b GT1b GM2 GQ1b .

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