Isolation and purification of acetylcholine receptor proteins by affinity chromatography.
In order to further molecular investigations on the binding capacity of acetylcholine receptors, a method was developed for the affinity chromatography of the nicotinic acetylcholine receptor. Reversibly binding cholinergic ligand groups were used as affinity ligands, instead of the well known snake venom alpha-toxins. These ligands are small in size, chemically well defined and fixed to long spacer chains (at least 40 nm). One ligand, with a pharmacologically stabilizing effect on the receptor, was a derivative of gallamine. Another, with a depolarizing effect, resembled carbamoylcholine and a third was a derivative of decamethonium. The receptor proteins were isolated from Torpedo marmorata electric organs. Preparation included solubilization with a non-ionic detergent, alkaline treatment to extract peripheral membrane proteins and affinity purification. The receptor proteins eluted from the three affinity resins were identical in their assembly of subunits (alpha, beta, gamma and delta) but of different purity. Receptor proteins were obtained on a large scale within a short time and under mild conditions for elution with the affinity ligands of the decamethonium or the gallamine type. This was a considerable advantage compared to the use of alpha-bungarotoxin.
Waser
P G
PG
Department of Pharmacology, University of Zürich, Switzerland.
Bodmer
D M
DM
Hopff
W H
WH
D000818:Animals; D002846:Chromatography, Affinity; D004557:Electric Organ / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D066298:In Vitro Techniques; D007457:Iodine Radioisotopes; D008024:Ligands; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000378:metabolism; D011506:Proteins / Q000302:isolation & purification*; D011950:Receptors, Cholinergic / Q000302:isolation & purification*; D012116:Resins, Plant; D013056:Spectrophotometry, Ultraviolet; D014101:Torpedo
D007457:Iodine Radioisotopes; D008024:Ligands; D009419:Nerve Tissue Proteins; D011506:Proteins; D011950:Receptors, Cholinergic; D012116:Resins, Plant