Incorporation of the nicotinic acetylcholine receptor into liposomes by rapid removal of detergent using poly (ethylene) glycol 6000.
The poly (ethylene) glycol 6000 (PEG) precipitation procedure was applied for incorporating the nicotinic acetylcholine receptor from D. tschudii electric organ into Asolectin liposomes. The incorporated receptor had its toxin binding sites oriented towards the external bulk phase and bound [(125)I]?-bungarotoxin with the same rate of toxin binding and cholinergic agonist equilibrium low affinity constant as its membrane bound counterpart. A characteristic feature of those vesicles, however, was the absence of the affinity transitions which normally correlate with the receptor densensitization phenomenon, and a poor carbamylcholine-stimulated ion flux as compared to that exhibited by vesicles prepared by the cholate dialysis procedure. When ether-purified PEG was used, the incorporated receptor showed affinity transitions and carbamylcholine-induced cation influx into vesicles. Thin layer chromatography and quantitative densitometry analysis showed that the vesicles prepared by the PEG precipitation method contained higher amounts of free fatty acids than those prepared by the cholate-dialysis procedure. Results are interpreted as an impairment of receptor function by impurities contained in commercial preparations of PEG and/or free fatty acids possibly generated by PEG impurities from Asolectin lipids. The proposed mechanisms are modifications in vesicle geometry and perturbations in lipid-protein interactions known to be essential for normal nicotinic receptor operation. The PEG precipitation procedure has the potential for rapid reconstitution of the nicotinic receptor and other membrane proteins that cannot be solubilized by easily dialyzable detergents provided purified PEG is used.
Medrano
S
S
Instituto de Biologia Celular, Facultad de Medicina, Universidad de Buenos Aires, 1121 Buenos Aires, Argentina.
Aguilar
J
J
Rivas
E
E
Ochoa
E L
EL