A new fluorimetric method to measure protein-catalyzed phospholipid transfer using 1-acyl-2-parinaroylphosphatidylcholine.
A new, simple and versatile method to measure phospholipid transfer has been developed, based on the use of a fluorescent phospholipid derivative, 1-acyl-2-parinaroylphosphatidylcholine. Vesicles prepared of this phospholipid show a low level of fluorescence due to interaction between the fluorescent groups. When phospholipid transfer protein and vesicles consisting of non-labeled phosphatidylcholine are added the protein catalyzes an exchange of phosphatidylcholine between the labeled donor and non-labeled acceptor vesicles. The insertion of labeled phosphatidylcholine into the non-labeled vesicles is accompanied by an increase in fluorescence due to abolishment of self-quenching. The initial rate of fluorescence enhancement was found to be proportional to the amount of transfer protein added. This assay was applied to determine the effect of membrane phospholipid composition on the activity of the phosphatidylcholine-, phosphatidylinositol- and non-specific phospholipid transfer proteins. Using acceptor vesicles of egg phosphatidylcholine and various amounts of phosphatidic acid it was observed that the rate of phosphatidylcholine transfer was either stimulated, inhibited or unaffected by increased negative charge depending on the donor to acceptor ratio and the protein used. In another set of experiments acceptor vesicles were prepared of phosphatidylcholine analogues in which the ester bonds were replaced with ether bonds or carbon-carbon bonds. Assuming that only a strictly coupled exchange between phosphatidylcholine and analogues gives rise to the observed fluorescence increase, orders of substrate preference should be established for the phosphatidylcholine- and phosphatidylinositol transfer proteins.
D002352:Carrier Proteins / Q000032:analysis*; D007700:Kinetics; D008081:Liposomes; D008244:Lysophosphatidylcholines; D008565:Membrane Proteins; D010713:Phosphatidylcholines; D049294:Phospholipid Transfer Proteins; D013050:Spectrometry, Fluorescence / Q000379:methods
D002352:Carrier Proteins; D008081:Liposomes; D008244:Lysophosphatidylcholines; D008565:Membrane Proteins; D010713:Phosphatidylcholines; D049294:Phospholipid Transfer Proteins; C030048:2-parinarolyllecithin