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Physiochemical and immunological properties of acetylcholine receptors from human muscle

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Summary

The acetylcholine receptor protein from human muscle was extracted with the non-ionic detergent Triton X-100 and purified by affinity chromatography on α-Naja toxin sepharose 4B. Further purification on Dicap-MP sepharose 4B, a choline analog compound, led to ACHR preparations with specific activities of 2–7 nmol/mg protein. The isolated receptor, labeled with 125I-α-bungarotoxin was characterized by different methods and compared to ACHRs from Torpedo californica electroplax and rat-denervated skeletal muscle. Gel filtration on Ultrogel AcA 34 resulted in a stokes radius of 70 Å for the receptor monomer and 99 Å for the dimeric form. Sucrose density gradient centrifugation showed sedimentation coefficients of 9.1 S and 13.5 S. From these data the molecular weight of the ACHR monomer was estimated as 254 000 D and 540 000 D for the receptor dimer. The isoelectric point of the 125I-α-bgt-ACHR complex was determined by thin-layer isoelectric focussing to be pH 5.

Purified ACHRs were used for immunization of rats and mice which developed an EAMG as verified by clinical observation and electrophysical measurements. Sera from the immunized animals as well as from myasthenia gravis patients were subsequently used to compare the cross-reactivity of ACHR preparations from different sources. While antibodies of rats immunized with Torpedo ACHRs cross-reacted with ACHR preparations from rat and human skeletal muscle, antibodies from mice immunized with rat ACHR only reacted with preparations from rats and mice. Antibodies from mice immunized with ACHR of human origin exhibited a broad cross-reactivity, as did antibodies from MG patients.

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Abbreviations

AB:

antibody

ACHR:

nicotinic acetylcholine receptor

BSA:

bovine serum albumin

Dicap-MP:

methyl-[N-(6-aminocaproyl-6′aminocaproyl)-3-amino]pyridinbromide

EAMG:

experimental autoimmune myasthenia gravis

EDTA:

ethylenediaminetetraaceticacid

MG:

myasthenia gravis

PMSF:

phenylmethylsulfonylfluoride

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Author information

Author notes

  1. Hartmut Wekerle

    Present address: Max-Planck-Gesellschaft klinische Forschungsgruppe MS, Würzburg

  2. Karl L. Birnberger

    Present address: Neurologische Klinik des Bezirks Niederbayern, Deggendorf

Authors and Affiliations

  1. Zentrum Biochemie der Medizinischen Hochschule Hannover, Karl-Wiechert-Allee, 3000, Hannover 61, Germany

    Inge Kalies & Fritz Heinz

  2. Max-Planck-Institut für Immunbiologie, Freiburg-Zehren, 7800, Freiburg, Germany

    Reinhard Hohlfeld & Hartmut Wekerle

  3. Neurologische Klinik der Technischen Universitat München, 8000, München, Germany

    Karl L. Birnberger

  4. Institut für klinische Immunologie und Rheumatologie der Universität Erlangen-Nürnberg, Krankenhausstr. 12, 8520, Erlangen, Germany

    Inge Kalies & Joachim R. Kalden

Authors
  1. Inge Kalies
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  2. Fritz Heinz
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  3. Reinhard Hohlfeld
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  4. Hartmut Wekerle
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  6. Joachim R. Kalden
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Additional information

Recipient of a postdoctoral grant from Deutsche Forschungsgemeinschaft; present address: Neurologische Klinik, Medizinische Einrichtungen der Universität Düsseldorf.

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Kalies, I., Heinz, F., Hohlfeld, R. et al. Physiochemical and immunological properties of acetylcholine receptors from human muscle. Mol Cell Biochem 64, 69–79 (1984). https://doi.org/10.1007/BF00420930

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  • DOI: https://doi.org/10.1007/BF00420930

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