Purification and biochemical characterization of nicotinic acetylcholine receptors of human muscle

M. Y. Momoi, V. A. Lennon

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31 Scopus citations

Abstract

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 α-neurotoxin. After elution with 1-2 M NaCl buffers from the immunoadsorbent and with carbamylcholine from the α-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 α-toxin-gel was 10%. Specific binding of α-bungarotoxin by the two purified preparations were, respectively, 5-6 pmol and 7 pmol/μg of protein, representing over 300,000-fold purification from the original detergent extract. Inoculation of 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(r)=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 bromoacetyl[3H]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, DEAE elution 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.

Original languageEnglish (US)
Pages (from-to)12757-12764
Number of pages8
JournalJournal of Biological Chemistry
Volume257
Issue number21
StatePublished - 1982

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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