Mattjus P, Molotkovsky J G, Smaby J M, Brown R E
Hormel Institute, University of Minnesota, Austin, Minnesota, 55912, USA.
Anal Biochem. 1999 Mar 15;268(2):297-304. doi: 10.1006/abio.1998.3065.
A lipid transfer protein, purified from bovine brain (23.7 kDa, 208 amino acids) and specific for glycolipids, has been used to develop a fluorescence resonance energy transfer assay (anthrylvinyl-labeled lipids; energy donors and perylenoyl-labeled lipids; energy acceptors) for monitoring the transfer of lipids between membranes. Small unilamellar vesicles composed of 1 mol% anthrylvinyl-galactosylceramide, 1.5 mol% perylenoyl-triglyceride, and 97.5% 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) served as donor membranes. Acceptor membranes were 100% POPC vesicles. Addition of glycolipid transfer protein to mixtures of donor and acceptor vesicles resulted in increasing emission intensity of anthrylvinyl-galactosylceramide and decreasing emission intensity of the nontransferable perylenoyl-triglyceride as a function of time. The behavior was consistent with anthrylvinyl-galactosylceramide being transferred from donor to acceptor vesicles. The anthrylvinyl and perylenoyl energy transfer pair offers advantages over frequently used energy transfer pairs such as NBD and rhodamine. The anthrylvinyl emission overlaps effectively the perylenoyl excitation spectrum and the fluorescence parameters of the anthrylvinyl fluorophore are nearly independent of the medium polarity. The nonpolar fluorophores are localized in the hydrophobic region of the bilayer thus producing minimal disturbance of the bilayer polar region. Our results indicate that this method is suitable for assay of lipid transfer proteins including mechanistic studies of transfer protein function.
一种从牛脑中纯化得到的（23.7 kDa，208个氨基酸）、对糖脂具有特异性的脂质转移蛋白，已被用于开发一种荧光共振能量转移测定法（蒽乙烯基标记的脂质；能量供体和苝酰基标记的脂质；能量受体），用于监测膜之间脂质的转移。由1 mol%蒽乙烯基 - 半乳糖神经酰胺、1.5 mol%苝酰基 - 甘油三酯和97.5% 1 - 棕榈酰 - 2 - 油酰磷脂酰胆碱（POPC）组成的小单层囊泡用作供体膜。受体膜是100% POPC囊泡。向供体和受体囊泡的混合物中添加糖脂转移蛋白，导致蒽乙烯基 - 半乳糖神经酰胺的发射强度随时间增加，而不可转移的苝酰基 - 甘油三酯的发射强度随时间降低。这种行为与蒽乙烯基 - 半乳糖神经酰胺从供体囊泡转移到受体囊泡一致。蒽乙烯基和苝酰基能量转移对相对于常用的能量转移对（如NBD和罗丹明）具有优势。蒽乙烯基发射有效地与苝酰基激发光谱重叠，并且蒽乙烯基荧光团的荧光参数几乎与介质极性无关。非极性荧光团位于双层的疏水区域，因此对双层极性区域的干扰最小。我们的结果表明，该方法适用于脂质转移蛋白的测定，包括转移蛋白功能的机制研究。
