Large‐Scale Purification of the Acetylcholine‐Receptor Protein in Its Membrane‐Bound and Detergent‐Extracted Forms from <i>Torpedo marmorata</i> Electric Organ

Abstract

A method is described for the large‐scale purification of membrane fragments very rich in acetylcholine (nicotinic) receptor from the electric organ of Torpedo marmorata . The preparations of purified membrane fragments have a specific activity of more than 4000 nmol α‐toxin binding sites/g protein and give only four main polypeptide bands by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Observations by electron microscopy show that the purified preparation of receptor‐rich membrane fragments is composed of only one class of membrane fragments covered with 8‐nm rosettes identified as acetylcholine receptor molecules. This preparation is used as a starting material for the detergent solubilization and the large‐scale purification of the acetylcholine receptor protein, without using affinity chromatography. A sucrose gradient centrifugation of a Triton X‐100 extract of receptor‐rich membranes done in the presence of 2‐mercaptoethanol yields large quantities of receptor protein in a homogeneous form as indicated by polyacrylamide gel electrophoresis, isoelectric focussing and electron microscopy. Polyacrylamide gel electrophoresis of the purified protein in the presence of sodium dodecylsulfate reveals three bands of apparent molecular weights 40000 ± 1000, 50000 ± 2000 and 66000 ± 2000, the two heaviest ones being present in significantly lower amounts than the 40000‐ M r one. The comparison of several samples of purified protein with different specific activities reveals a striking variability of the ratios of the 40000‐ M r band to the 50000 and 66000‐ M r ones.