Purification and biochemical characterization of nicotinic acetylcholine receptors of human muscle.

Nicotinic acetylcholine receptors (AChR) extracted from skeletal muscle of amputated human limbs in 2% Triton X-100 were purified by two different methods of affinity chromatography, agarose conjugated with monoclonal anti-AChR antibody or a-neurotoxin. After elution, with 1-2 M NaCl buffers from the immunoadsorbent and with carbamylcholine from the a-toxin gel, the preparations were purified further by chromatography on DEAE-Sephadex A-50 with NaCl gradient elution. Final recovery of AChR from the immunoadsorbent was 20-30% and from a-toxin-gel was 10%. Specific binding of a-bungarotoxin by the two purified preparations were, respectively, 5-6 pmol and 7 pmol/ pg of protein, representing over 300,000-fold purification from the original detergent extract. Inoculation f a rat with 49 pmol in adjuvants induced severe experimental autoimmune myasthenia gravis. After electrophoresis under reducing conditions on 10% sodium dodecyl sulfate-polyacrylamide gels, silver staining of AChR from muscle without evidence of vascular or neuromuscular disease revealed five predominant polypeptides: M, = 44,000, 53,000, 56,000, 61,000, and 66,000. Human muscle AChR in Triton X-100 existed as a 9 S protein with an isoelectric point of 5.0. Reaction with the affinity labeling reagent br~moacetyl[~H]choline bromide revealed a single peak of cholinergic ligand binding associated with the 44,000-dalton polypeptide. Muscle with occlusive vascular disease yielded 9 times more AChR than muscle without vascular disease, ( p < 0.025), and AChR from ischemic muscle was heterogeneous in isoelectric focusing, DEelution profile, and sucrose gradient centrifugation (additional peaks smaller than 9 S). Thus the presence of ischemia introduces a significant factor of variability in biochemical analyses of muscle AChR.