| 1. |
- Karlsson, Margareta, et al.
(författare)
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Lipid composition of caveolae and of surrounding plasma membrane in rat adipocytes
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Caveolae are invaginations of the plasma membrane that may arise from so called rafts in the presence of the structural protein caveolin. We have isolated caveolae from purified plasma membrane of primary rat adipocytes using ultrasonication to disrupt the membrane followed by density gradient ultracentrifugation. This caveolae fraction was further purified by adsorption to antibodies against caveolin. As a comparison we also isolated a detergent-insoluble fraction of the plasma membrane, utilizing the detergent insolubility of caveolae and rafts. Caveolae were strongly enriched in cholesterol and sphingomyelin, the concentration was 3.5 and 2.8-fold, respectively, higher in the caveolar membrane than in the surrounding plasma membrane. Phosphoacylglycerols were also concentrated in caveolae, while proteins were depleted compared to the surrounding plasma membrane. We have calculated that an average adipocyte caveola contains 18000 molecules of cholesterol, 6000 of sphingomyelin, 18000 of phosphoacylglycerol, 350 protein molecules, and about I 00 glycolipid molecules.We analyzed for a range of glycolipids and especially gangliosides. Of these GM3 and GD3 are the most prevalent and both were enriched in caveolae, together with GM1 and GDla. GDlb and GTib were present in the plasma membrane at low levels, while GM2, GD2, GQ1b, sulphatide, and lactosylceramide sulphate were not detected. None of them were detected in caveolae. As a first comprehensive and quantitative analysis of purified caveolae from primary cells, our results provide a firm basis for the examination of caveolae formation using artificial membranes.
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| 2. |
- Örtegren, Unn, 1975-, et al.
(författare)
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Lipids and glycosphingolipids in caveolae and surrounding plasma membrane of primary rat adipocytes
- 2004
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Ingår i: Eur J Biochem. - : Wiley. - 0014-2956 .- 1432-1033. ; 271:10, s. 2028-36
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Tidskriftsartikel (refereegranskat)abstract
- 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 glycerophospholipids 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 10(3) molecules of cholesterol, 7.5 x 10(3) of sphingomyelin and 23 x 10(3) 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, sulfatide and lactosylceramide sulfate were not detected in caveolae.
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