The stability of solubilized mammalian muscle acetylcholine receptors during purification by monoclonal immunoadsorption

M. Y. Momoi, Vanda A Lennon

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Abstract

The stability of nicotinic acetylcholine receptors (AChR) solubilized from mammalian skeletal muscle in nonionic detergent was investigated under various conditions of pH, chaotropic ions, and unfolding reagents in order to allow its purification in high yield by immunoadsorption to monoclonal antibodies. Preservation of the antigenicity and/or binding sites for α-bungarotoxin was used as an indicator of the receptor protein's integrity. Both were preserved in the pH range 6.5-8.0, but when exposed for 1 h at 4°C to a pH outside this range, >50% activity was lost. Of the chaotropic ions studied (NaSCN, NaI, NaNO3, NaCl), only NaCl was tolerated. Most of the AChR's toxin-binding activity was preserved after exposure to 2 M NaCl, which was suitable for dissociating AChR when a monoclonal antibody with relatively low binding affinity was selected as the immunoadsorbent. Yields of purified AChR were optimal (30%) when a low amount of monoclonal antibody was coupled to cyanogen bromide-activated agarose (1 mg protein/ml gel).

Original languageEnglish (US)
Pages (from-to)59-64
Number of pages6
JournalJournal of Neurochemistry
Volume42
Issue number1
StatePublished - 1984

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Cholinergic Receptors
Purification
Muscle
Monoclonal Antibodies
Muscles
Ions
Bungarotoxins
Cyanogen Bromide
Immunosorbents
Nicotinic Receptors
Detergents
Sepharose
Skeletal Muscle
Proteins
Gels
Binding Sites

ASJC Scopus subject areas

  • Biochemistry
  • Cellular and Molecular Neuroscience

Cite this

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title = "The stability of solubilized mammalian muscle acetylcholine receptors during purification by monoclonal immunoadsorption",
abstract = "The stability of nicotinic acetylcholine receptors (AChR) solubilized from mammalian skeletal muscle in nonionic detergent was investigated under various conditions of pH, chaotropic ions, and unfolding reagents in order to allow its purification in high yield by immunoadsorption to monoclonal antibodies. Preservation of the antigenicity and/or binding sites for α-bungarotoxin was used as an indicator of the receptor protein's integrity. Both were preserved in the pH range 6.5-8.0, but when exposed for 1 h at 4°C to a pH outside this range, >50{\%} activity was lost. Of the chaotropic ions studied (NaSCN, NaI, NaNO3, NaCl), only NaCl was tolerated. Most of the AChR's toxin-binding activity was preserved after exposure to 2 M NaCl, which was suitable for dissociating AChR when a monoclonal antibody with relatively low binding affinity was selected as the immunoadsorbent. Yields of purified AChR were optimal (30{\%}) when a low amount of monoclonal antibody was coupled to cyanogen bromide-activated agarose (1 mg protein/ml gel).",
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T1 - The stability of solubilized mammalian muscle acetylcholine receptors during purification by monoclonal immunoadsorption

AU - Momoi, M. Y.

AU - Lennon, Vanda A

PY - 1984

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N2 - The stability of nicotinic acetylcholine receptors (AChR) solubilized from mammalian skeletal muscle in nonionic detergent was investigated under various conditions of pH, chaotropic ions, and unfolding reagents in order to allow its purification in high yield by immunoadsorption to monoclonal antibodies. Preservation of the antigenicity and/or binding sites for α-bungarotoxin was used as an indicator of the receptor protein's integrity. Both were preserved in the pH range 6.5-8.0, but when exposed for 1 h at 4°C to a pH outside this range, >50% activity was lost. Of the chaotropic ions studied (NaSCN, NaI, NaNO3, NaCl), only NaCl was tolerated. Most of the AChR's toxin-binding activity was preserved after exposure to 2 M NaCl, which was suitable for dissociating AChR when a monoclonal antibody with relatively low binding affinity was selected as the immunoadsorbent. Yields of purified AChR were optimal (30%) when a low amount of monoclonal antibody was coupled to cyanogen bromide-activated agarose (1 mg protein/ml gel).

AB - The stability of nicotinic acetylcholine receptors (AChR) solubilized from mammalian skeletal muscle in nonionic detergent was investigated under various conditions of pH, chaotropic ions, and unfolding reagents in order to allow its purification in high yield by immunoadsorption to monoclonal antibodies. Preservation of the antigenicity and/or binding sites for α-bungarotoxin was used as an indicator of the receptor protein's integrity. Both were preserved in the pH range 6.5-8.0, but when exposed for 1 h at 4°C to a pH outside this range, >50% activity was lost. Of the chaotropic ions studied (NaSCN, NaI, NaNO3, NaCl), only NaCl was tolerated. Most of the AChR's toxin-binding activity was preserved after exposure to 2 M NaCl, which was suitable for dissociating AChR when a monoclonal antibody with relatively low binding affinity was selected as the immunoadsorbent. Yields of purified AChR were optimal (30%) when a low amount of monoclonal antibody was coupled to cyanogen bromide-activated agarose (1 mg protein/ml gel).

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