Detection of protein mediated glycosphingolipid clustering by the use of resonance energy transfer between fluorescent labelled lipids. A method established by applying the system ganglioside GM1 and cholera toxin B subunit
✍ Scribed by Peter Antes; Günter Schwarzmann; Konrad Sandhoff
- Publisher
- Elsevier Science
- Year
- 1992
- Tongue
- English
- Weight
- 791 KB
- Volume
- 62
- Category
- Article
- ISSN
- 0009-3084
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✦ Synopsis
Glycosphingolipids labelled in the ceramide moiety with 3-(p-(6-phenyl)-l,3,5-hexatrienyl)phenylpropionic acid (DPH) or 6-(4nitrobenz-2-oxa-1,3-diazole-7-yl)aminohexanoic acid (NBD) were incorporated into small unilamellar lecithin liposomes. They were used in resonance energy transfer (RET) experiments between the donor fluorophore DPH and the acceptor NBD to study glycosphingolipid distribution. In pure lecithin liposomes the fluorescent derivatives of GM 1, GAI, galactosylceramide and sulfatide behaved almost identically and Ca 2+ ions (5 I~M or 150 mM) did not influence their transfer efficiencies. But cholera toxin B subunit (CTB) specifically clustered GM1 and enhanced the transfer efficiency. This RET-based method facilitated determination of binding specificity, complex stoichiometry (CTB/GM 1 = 1:5), half time of complex formation (5 s), cooperativity in binding and had a maximal sensitivity at a liposome dotation rate of just 0.25 mol%. In contrast to this, anisotropy of the fluorophores and the excimer to monomer ratio of pyrene-GMl were not affected by CTB. This demonstrates the advantage of the presented technique in detection of protein mediated glycosphingolipid clustering.