Purification and characterization of nicotinic acetylcholine receptors from muscle.

The nicotinic acetylcholine receptor was purified from normal and denervated rat skeletal muscle. The purification protocol included alpha-cobratoxin biospecific adsorption, ion exchange chromatography, and gel filtration steps. The highest specific activity achieved was 7.5 pmol of 125I-alpha-bungarotoxin binding sites per microgram protein. Sodium dodecyl sulfate gel electrophoresis of purified AChR revealed subunits with molecular weights of 42,000 and 66,000 daltons and a minor component with a molecular weight of 52,000 daltons. Normal muscle AChR is comprised of one toxin binding component. Upon denervation a second component appears, but both components are increased as a consequence of denervation. A dissociation constant of 1.5 x 10(-8)M was determined for d-tubocurarine from receptor from both normal and denervated muscle. A dissociation constant of 1 x 10(-7)M for acetylcholine, perhaps analogous to the high affinity acetylcholine binding observed in electric fish receptor, was determined.