Binding of fluorescent-labeled phosphatidylcholine to rat liver nonspecific lipid transfer protein.
The fluorescent-labeled phospholipid, 1-palmitoyl-2-[12-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]dodecan oyl] phosphatidylcholine (P-C12-NBD-PC), is highly self-quenched when contained as a high mole percent in phospholipid vesicles. The addition of nonspecific lipid transfer protein (nsLTP), which is identical to sterol carrier protein2, to P-C12-NBD-PC self-quenching vesicles results in a large increase in the fluorescence yield. Evidence is presented that demonstrates that this fluorescence increase results from the binding of P-C12-NBD-PC to one or more low dielectric sites on the nsLTP molecule and that the P-C12-NBD-PC.nsLTP complex can exist free in aqueous solution. The binding is inhibited in the presence of mersalyl, a thiol reagent, and can be reversed with excess cysteine. Transfer of the nsLTP-bound P-C12-NBD-PC to phospholipid vesicles is also demonstrated. The half-times for loading and unloading the P-C12-NBD-PC.nsLTP complex are both 3 orders of magnitude faster than the spontaneous P-C12-NBD-PC transfer between vesicles. These data demonstrate that nsLTP has the necessary properties to bind and carry lipids between membranes.
Nichols
J W
JW
Department of Physiology, Emory University School of Medicine, Atlanta, Georgia 30322.
D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives / Q000378:metabolism*; D000818:Animals; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D005456:Fluorescent Dyes; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D008634:Mersalyl / Q000494:pharmacology; D010069:Oxadiazoles / Q000378:metabolism*; D010713:Phosphatidylcholines / Q000378:metabolism*; D051381:Rats; D013050:Spectrometry, Fluorescence
D002352:Carrier Proteins; D005456:Fluorescent Dyes; D010069:Oxadiazoles; D010713:Phosphatidylcholines; C030363:lipid transfer protein; C053903:1-palmitoyl-2-(12-((7-nitro-2,1,3-benzoxadiazol-4-yl)amino)dodecanoyl)phosphatidylcholine; D008634:Mersalyl; D009327:4-Chloro-7-nitrobenzofurazan