Lipid composition of caveolae and of surrounding plasma membrane in rat adipocytes
(English)Manuscript (preprint) (Other academic)
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.
Medical and Health Sciences
IdentifiersURN: urn:nbn:se:liu:diva-84464OAI: oai:DiVA.org:liu-84464DiVA: diva2:559411