Chemical transduction with fluorescent lipid membranes using selective interactions of acetylcholine receptor with agonist/antagonist and acetylcholine with substrate.
Alterations in the physical structure of vesicles and monolayers of phospholipids and soybean lecithin were monitored by measurement on the average fluorescence intensity changes from N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)dipalmitoyl-L-a-phosphatidyl ethanolamine (NBD-PE) located in the lipid matrices. This probe was intimately dispersed at a concentration of 1-2 mol-% in lipid membranes and had an emission sensitive to local environmental structure. Alterations in the structure of soybean lecithin vesicles were induced by the selective interaction of acetylcholine receptor with the agonist carbamylcholine and the antagonist alpha-bungarotoxin. Structural changes in vesicles with a 7:3 mole ratio of dipalmitoylphosphatidyl choline to dipalmitoylphosphatidic acid were observed for selective interactions between acetylcholinesterase and acetylcholine. Enhancement of fluorescence emission from the lipid membranes provided transduction of the selective binding events of the receptor and enzyme. A maximum sensitivity of about a 30% enhancement per micromole of carbamylcholine and a detection limit for the toxin of 10 nM were observed for the receptor. Fluorescence microscopy was used to establish that protein could be incorporated in monolayer lipid membranes and to provide information about potential mechanisms of fluorescence enhancement. These studies show that lipid membranes containing NBD-PE can be used as generic transducers of protein-ligand interactions.
Krull
U J
UJ
Chemical Sensors Group, Departmentof Chemistry, University of Toronto, Mississauga, Ontairo, Canada.
Brennan
J D
JD
Brown
R S
RS
Hosein
S
S
Hougham
B D
BD
Vandenberg
E T
ET
D000109:Acetylcholine / Q000378:metabolism; D000110:Acetylcholinesterase / Q000378:metabolism*; D015374:Biosensing Techniques; D002038:Bungarotoxins / Q000378:metabolism*; D002217:Carbachol / Q000378:metabolism*; D005456:Fluorescent Dyes; D008051:Lipid Bilayers / Q000378:metabolism; D008567:Membranes, Artificial; D008856:Microscopy, Fluorescence; D010713:Phosphatidylcholines; D010743:Phospholipids; D011950:Receptors, Cholinergic / Q000378:metabolism*
D002038:Bungarotoxins; D005456:Fluorescent Dyes; D008051:Lipid Bilayers; D008567:Membranes, Artificial; D010713:Phosphatidylcholines; D010743:Phospholipids; D011950:Receptors, Cholinergic; D002217:Carbachol; D000110:Acetylcholinesterase; D000109:Acetylcholine