The Affinity-Labeling of Partially Purified Acetylcholine Receptor from Electric Tissue of Electrophorus
- 1 December 1973
- journal article
- Published by Proceedings of the National Academy of Sciences in Proceedings of the National Academy of Sciences
- Vol. 70 (12) , 3636-3640
- https://doi.org/10.1073/pnas.70.12.3636
Abstract
The receptor for acetylcholine was partially purified by affinity chromatography of an extract in Triton X-100 of membrane fragments from electric tissue. The receptor was assayed, after its reduction with dithiothreitol, by reaction with the affinity-alkylating agent, [methyl-(3)H]4-(N-maleimido)-benzyltrimethylammonium iodide. Alternative labeling procedures, one useful for routine assay of picomole quantities of receptor and the other for labeling larger quantities of receptor, are described. The purified receptor specifically incorporated about 3 nmol of label per mg of protein. This incorporation was blocked by pretreatment of the receptor with Naja naja siamensis neurotoxin. The rate of the affinity reaction was similar to that found with membrane fragments and with intact electroplax. Furthermore, as in intact electroplax, [(3)H]4-(N-maleimido)-benzyltrimethylammonium iodide reacted 5000-fold faster with the reduced receptor than did [(14)C]N-ethylmaleimide. When purified receptor was labeled with [(3)H]4-(N-maleimido)-benzyltrimethylammonium iodide and subjected to electrophoresis on polyacrylamide gels in dodecyl sulfate and dithiothreitol, three major protein bands were observed. Only one of these, however, contained (3)H activity; its mobility indicated a molecular weight of 40,000.Keywords
This publication has 22 references indexed in Scilit:
- Fluorescamine: A Reagent for Assay of Amino Acids, Peptides, Proteins, and Primary Amines in the Picomole RangeScience, 1972
- Isolation of the nicotinic acetylcholine receptor by biospecific chromatography on insolubilized Naja naja neurotoxinFEBS Letters, 1972
- Studies of the binding of α‐bungarotoxin to membrane‐bound and detergent‐dispersed acetylcholine receptors from Torpedo electric tissueFEBS Letters, 1972
- Progress in the purification of the cholinergic receptor protein from Electrophorus electricus by affinity chromatographyFEBS Letters, 1972
- Properties of the nicotinic acetylcholine receptor macromolecule of Electrophorus electricusFEBS Letters, 1972
- Affinity labeling of the acetylcholine receptor in the electroplaxJournal of Molecular Biology, 1971
- Electrophoretic analysis of the major polypeptides of the human erythrocyte membraneBiochemistry, 1971
- Affinity oxidation of the reduced acetylcholine receptorBiochimica et Biophysica Acta (BBA) - Biomembranes, 1970
- Elapid Neurotoxins and Their Mode of ActionClinical Toxicology, 1970
- Acetylcholine Receptor: Covalent Attachment of Depolarizing Groups at the Active SiteScience, 1969