The nicotinic acetylcholine regulator of Torpedo californica : purification and reconstruction

Acetylcholine receptor was extracted from the electric tissue of Torpedo californica by sodium cholate. The purification was performed by using density gradient centrifugation instead of affinity chromatography. The purified receptor was then incorporated into lipid vesicles by gel filtration on Sephadex G-50. The reconstituted vesicles were unilamellar, as revealed by negative staining electron microscopy. The vesicles were heterogeneous in size with an average diameter of 0.39+0.18 µm. Approximately 50% of the receptor was oriented towards the outside in these vesicles. Sodium dodecyl sulphate gel electrophoresis showed four bands of apparent molecular weight of 17,000, 40,000, 60,000, and 90,000 daltons from purified receptor either before or after reconstitution.

The function of incorporated acetylcholine receptors was tested by measurement of Na influx into the reconstituted vesicles. The influx was increased in the presence of O.02mM carbamylcholine, a cholinergic agonist of the receptor. This excitability was blocked by preincubation with 2mM d-tubocurarine, an antagonist of the receptor.

These results, therefore, demonstrated that not only the specific neurotransmitter binding sites but also the molecular elements necessary for ion translocation were reconstituted into the artificial lipid vesicles.