Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1987 Sep;84(18):6587–6590. doi: 10.1073/pnas.84.18.6587

Characterization of a purified nicotinic receptor from rat brain by using idiotypic and anti-idiotypic antibodies.

L G Abood, J J Langone, R Bjercke, X Lu, S Banerjee
PMCID: PMC299124  PMID: 3498166

Abstract

The availability of an anti-nicotine monoclonal antibody has made it possible to further establish the nature of the nicotine recognition proteins purified from rat brain by affinity chromatography and to provide a highly sensitive assay for determining [3H]nicotine binding to the purified material. An enantiomeric analogue of nicotine, (-)-6-hydroxymethyl-nicotine, was used to prepare the affinity column. In addition, with the use of anti-idiotypic monoclonal antibody, it was confirmed that the recognition site for nicotine resides on a protein complex composed of two components with molecular masses of 62 and 57 kDa. It was also demonstrated that the same two proteins could be purified by immunoaffinity chromatography with the use of an anti-idiotypic monoclonal antibody. With the use of the anti-nicotine antibody to measure [3H]nicotine binding, the purified material was shown to bind 250 pmol/mg of protein. By utilizing a procedure in which the purified receptor protein was conjugated to membranes by disulfide bonds, a binding activity of 80 pmol/mg was obtained. With the availability of stereospecific monoclonal antibodies to (-)-nicotine as well as monoclonal anti-idiotypic antibodies derived when the anti-nicotine antibodies were used as immunogens, additional procedures became available for the further characterization of the purified nicotine receptor and examining its (-)-[3H]nicotine-binding characteristics.

Full text

PDF
6587

Images in this article

6589Image
on p.6589

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Abood L. G., Reynolds D. T., Booth H., Bidlack J. M. Sites and mechanisms for nicotine's action in the brain. Neurosci Biobehav Rev. 1981 Winter;5(4):479–486. doi: 10.1016/0149-7634(81)90018-x. [DOI] [PubMed] [Google Scholar]
  2. Bjercke R. J., Cook G., Rychlik N., Gjika H. B., Van Vunakis H., Langone J. J. Stereospecific monoclonal antibodies to nicotine and cotinine and their use in enzyme-linked immunosorbent assays. J Immunol Methods. 1986 Jun 24;90(2):203–213. doi: 10.1016/0022-1759(86)90077-3. [DOI] [PubMed] [Google Scholar]
  3. Block G. A., Billiar R. B. Immunologic similarities between the hypothalamic alpha-bungarotoxin receptor and the Torpedo californica nicotinic cholinergic receptor. Brain Res. 1979 Dec 14;178(2-3):381–387. doi: 10.1016/0006-8993(79)90700-5. [DOI] [PubMed] [Google Scholar]
  4. Conti-Tronconi B. M., Dunn S. M., Barnard E. A., Dolly J. O., Lai F. A., Ray N., Raftery M. A. Brain and muscle nicotinic acetylcholine receptors are different but homologous proteins. Proc Natl Acad Sci U S A. 1985 Aug;82(15):5208–5212. doi: 10.1073/pnas.82.15.5208. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Conti-Tronconi B. M., Raftery M. A. The nicotinic cholinergic receptor: correlation of molecular structure with functional properties. Annu Rev Biochem. 1982;51:491–530. doi: 10.1146/annurev.bi.51.070182.002423. [DOI] [PubMed] [Google Scholar]
  6. Froehner S. C., Reiness C. G., Hall Z. W. Subunit structure of the acetylcholine receptor from denervated rat skeletal muscle. J Biol Chem. 1977 Dec 10;252(23):8589–8596. [PubMed] [Google Scholar]
  7. Gershoni J. M., Hawrot E., Lentz T. L. Binding of alpha-bungarotoxin to isolated alpha subunit of the acetylcholine receptor of Torpedo californica: quantitative analysis with protein blots. Proc Natl Acad Sci U S A. 1983 Aug;80(16):4973–4977. doi: 10.1073/pnas.80.16.4973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Homcy C. J., Rockson S. G., Haber E. An antiidiotypic antibody that recognizes the beta-adrenergic receptor. J Clin Invest. 1982 May;69(5):1147–1154. doi: 10.1172/JCI110550. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Jacob M. H., Berg D. K. The ultrastructural localization of alpha-bungarotoxin binding sites in relation to synapses on chick ciliary ganglion neurons. J Neurosci. 1983 Feb;3(2):260–271. doi: 10.1523/JNEUROSCI.03-02-00260.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Laemmli U. K., Favre M. Maturation of the head of bacteriophage T4. I. DNA packaging events. J Mol Biol. 1973 Nov 15;80(4):575–599. doi: 10.1016/0022-2836(73)90198-8. [DOI] [PubMed] [Google Scholar]
  11. Miledi R., Szczepaniak A. C. Effect of Dendroaspis neurotoxins on synaptic transmission in the spinal cord of the frog. Proc R Soc Lond B Biol Sci. 1975 Jul 1;190(1099):267–274. doi: 10.1098/rspb.1975.0092. [DOI] [PubMed] [Google Scholar]
  12. Olsen R. W., Meunier J. C., Changeux J. P. Progress in the purification of the cholinergic receptor protein from Electrophorus electricus by affinity chromatography. FEBS Lett. 1972 Nov 15;28(1):96–100. doi: 10.1016/0014-5793(72)80686-0. [DOI] [PubMed] [Google Scholar]
  13. Reilly T. M., Root R. T. Production of idiotypic and anti-idiotypic antibodies by BALB/c mice in response to immunizations with glucagon, vasopressin, or insulin: supporting evidence for the network concept. J Immunol. 1986 Jul 15;137(2):597–602. [PubMed] [Google Scholar]
  14. Schmidt J. T., Freeman J. A. Electrophysiologic evidence that retinotectal synaptic transmission in the goldfish is nicotinic cholinergic. Brain Res. 1980 Apr 7;187(1):129–142. doi: 10.1016/0006-8993(80)90499-0. [DOI] [PubMed] [Google Scholar]
  15. Schmidt J., Raftery M. A. Use of affinity chromatography for acetylcholine receptor purification. Biochem Biophys Res Commun. 1972 Oct 17;49(2):572–578. doi: 10.1016/0006-291x(72)90449-4. [DOI] [PubMed] [Google Scholar]
  16. Sloan J. W., Todd G. D., Martin W. R. Nature of nicotine binding to rat brain P2 fraction. Pharmacol Biochem Behav. 1984 Jun;20(6):899–909. doi: 10.1016/0091-3057(84)90015-7. [DOI] [PubMed] [Google Scholar]
  17. Smith M. A., Margiotta J. F., Franco A., Jr, Lindstrom J. M., Berg D. K. Cholinergic modulation of an acetylcholine receptor-like antigen on the surface of chick ciliary ganglion neurons in cell culture. J Neurosci. 1986 Apr;6(4):946–953. doi: 10.1523/JNEUROSCI.06-04-00946.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Whiting P. J., Lindstrom J. M. Purification and characterization of a nicotinic acetylcholine receptor from chick brain. Biochemistry. 1986 Apr 22;25(8):2082–2093. doi: 10.1021/bi00356a037. [DOI] [PubMed] [Google Scholar]
  20. Whiting P., Lindstrom J. Purification and characterization of a nicotinic acetylcholine receptor from rat brain. Proc Natl Acad Sci U S A. 1987 Jan;84(2):595–599. doi: 10.1073/pnas.84.2.595. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES