Purification and Characterization of Nicotinic Acetylcholine Receptors from Muscle

Abstract

The nicotinic acetylcholine receptor was purified from normal and denervated rat skeletal muscle. The purification protocol included α-cobratoxin biospecific adsorption, ion exchange chromatography, and gel filtration steps. The highest specific activity achieved was 7.5 pmol of ¹²⁵I-α-bungarotoxin binding sites per μg 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 × 10^-8M was determined for dtubocurarine from receptor from both normal and denervated muscle. A dissociation constant of 1 × 10^-7M for acetylcholine, perhaps analogous to the high affinity acetylcholine binding observed in electric fish receptor, was determined.