Use of fluorimetric method to assay the binding and transfer of phospholipids by lipid transfer proteins from maize seedlings and Arabidopsis leaves
1994
Moreau, F. | Virville, J.D. de | Hoffelt, M. | Guerbette, F. | Kader, J.C.
A fluorimetric method has been used to study the binding and transfer of phospholipids mediated by a lipid transfer protein (LTP) from maize seedlings and by proteins extracted from Arabidopsis leaves. This method is based on the use of donor vesicles prepared by sonication or injection and consisting of either self quenching vesicles of (1-palmitoyl 2-[12-[(7-nitrobenzoxadiazol-4-yl)amino] dodecanoyl]-Sn-glycero-3-phosphocholine(NBD-PC) or trinitrophenylphosphate phosphatidyl ethanolamine (TNP-PE) quenched vesicles of 1-palmitoyl-2(1-pyredecanoyl)-Sn-glycero-3-phosphocholine (Pyr-PC). Acceptor vesicles consisted in a mixture (90/10; v/v) of dioleoylphosphatidylcholine and phosphatidyl inositol (DOPC-PI). The use of injected vesicles of Pyr-PC allowed to achieve sensitive and qualitative tests of binding and transfer since 0.05 micromolar LTP were detected. By contrast, when sonicated vesicles of NBD-PC were used, quantitative determinations of phospholipid-LTP complex as well as measurement of the initial rate of phospholipid transfer on a large range of protein concentration (0.5 to 20 micromolar) were performed. This fluorimetric method has been successfully used to study the activity of maize LTP during its purification or the activity of LTPs present in Arabidopsis extracts.
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