Spontaneous, intervesicular transfer rates of fluorescent, acyl chain-labeled phosphatidylcholine analogs.
It was recently shown that the structure of the fluorophore attached to the acyl chain of phosphatidylcholine analogs determines their mechanism of transport across the plasma membrane of yeast cells (Elvington et al., J. Biol Chem. 280:40957, 2005). In order to gain further insight into the physical properties of these fluorescent phosphatidylcholine (PC) analogs, the rate and mechanism of their intervesicular transport was determined. The rate of spontaneous exchange was measured for PC analogs containing either NBD (7-nitrobenz-2-oxa-1,3-diazol-4-yl), Bodipy FL (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene), Bodipy 530 (4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene), or Bodipy 581 (4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene) attached to a five or six carbon acyl chain in the sn-2 position. The rate of transfer between phospholipid vesicles was measured by monitoring the increase in fluorescence as the analogs transferred from donor vesicles containing self-quenching concentrations to unlabeled acceptor vesicles. Kinetic analysis indicated that the transfer of each analog occurred by diffusion through the water phase as opposed to transfer during vesicle collisions. The vesicle-to-monomer dissociation rate constants differed by over four orders of magnitude: NBD-PC (k(dis)=0.115 s(-1); t(1/2)=6.03 s); Bodipy FL-PC (k(dis)=5.2x10(-4); t(1/2)=22.2 min); Bodipy 530-PC (k(dis)=1.52x10(-5); t(1/2)=12.6 h); and Bodipy 581-PC (k(dis)=5.9x10(-6); t(1/2)=32.6 h). The large differences in spontaneous rates of transfer through the water measured for these four fluorescent PC analogs reflect their hydrophobicity and may account for their recognition by different mechanisms of transport across the plasma membrane of yeast.
Elvington
Shelley M
SM
Department of Physiology, 605G Whitehead Building, 615 Michael Street, Emory University School of Medicine, Atlanta, GA 30322, USA.
Nichols
J Wylie
JW
D001393:Azoles; D005456:Fluorescent Dyes; D005470:Fluorometry; D007700:Kinetics; D015394:Molecular Structure; D009578:Nitrobenzenes; D010713:Phosphatidylcholines / Q000737:chemistry / Q000378:metabolism*
C510797:7-nitrobenz-2-oxa-1,3-diazol-4-yl; D001393:Azoles; D005456:Fluorescent Dyes; D009578:Nitrobenzenes; D010713:Phosphatidylcholines; C028694:1-palmitoyl-2-oleoylphosphatidylcholine