Skip to main content
NCBI home page
Biophysical Journal logoLink to Biophysical Journal
. 1984 Feb;45(2):447–454. doi: 10.1016/S0006-3495(84)84167-3

Kinetics of interaction of N epsilon-fluorescein isothiocyanate-lysine-23-cobra alpha-toxin with the acetylcholine receptor.

A T Cheung, D A Johnson, P Taylor
PMCID: PMC1434853  PMID: 6696970

Abstract

We have studied the kinetics of N epsilon-fluorescein isothiocyanate-lysine-23 cobra alpha-toxin (FITC-toxin) binding to the membrane-associated acetylcholine receptor from the Torpedo californica electric organ. The fluorescent toxin not only enabled us to monitor the binding reaction continuously but also to examine simultaneously the enhancement of ligand fluorescence and the increase in steady state polarization of fluorescence associated with binding of the alpha-toxin. Over the range of concentrations employed, both parameters yielded identical kinetic constants, suggesting that the enhancement of fluorescence of fluorescein and its immobilization are occurring in the same time frame. Both an initial rate analysis and the integrated rate expression showed association to be a simple, reversible bimolecular process. The apparent second-order association rate constant derived from the integrated rate analysis was constant within a factor of 2 over a 40-fold concentration range (6.7 +/- 1.7 X 10(3) M-1 S-1). The unimolecular dissociation rate constant was found to be 3.3 +/- 0.5 X 10(-5) S-1.

Full text

PDF
447

Selected References

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

  1. Blanchard S. G., Quast U., Reed K., Lee T., Schimerlik M. I., Vandlen R., Claudio T., Strader C. D., Moore H. P., Raftery M. A. Interaction of [125I]-alpha-bungarotoxin with acetylcholine receptor from Torpedo californica. Biochemistry. 1979 May 15;18(10):1875–1883. doi: 10.1021/bi00577a005. [DOI] [PubMed] [Google Scholar]
  2. CHANG C. C., LEE C. Y. ISOLATION OF NEUROTOXINS FROM THE VENOM OF BUNGARUS MULTICINCTUS AND THEIR MODES OF NEUROMUSCULAR BLOCKING ACTION. Arch Int Pharmacodyn Ther. 1963 Jul 1;144:241–257. [PubMed] [Google Scholar]
  3. Changeux J. P., Kasai M., Lee C. Y. Use of a snake venom toxin to characterize the cholinergic receptor protein. Proc Natl Acad Sci U S A. 1970 Nov;67(3):1241–1247. doi: 10.1073/pnas.67.3.1241. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Changeux J. P. The acetylcholine receptor: an "allosteric" membrane protein. Harvey Lect. 1979 1980;75:85–254. [PubMed] [Google Scholar]
  5. Dandliker W. B., Levison S. A. Investigation of antigen-antibody kinetics by fluorescence polarization. Immunochemistry. 1968 Mar;5(2):171–183. doi: 10.1016/0019-2791(68)90101-8. [DOI] [PubMed] [Google Scholar]
  6. Johnson D. A., Taylor P. Site-specific fluorescein-labeled cobra alpha-toxin. Biochemical and spectroscopic characterization. J Biol Chem. 1982 May 25;257(10):5632–5636. [PubMed] [Google Scholar]
  7. Kang S., Maelicke A. Fluorescein isothiocyanate-labeled alpha-cobratoxin. Biochemical characterization and interaction with acetylcholine receptor from Electrophorus electricus. J Biol Chem. 1980 Aug 10;255(15):7326–7332. [PubMed] [Google Scholar]
  8. Karlsson E., Arnberg H., Eaker D. Isolation of the principal neurotoxins of two Naja naja subspecies. Eur J Biochem. 1971 Jul 15;21(1):1–16. doi: 10.1111/j.1432-1033.1971.tb01433.x. [DOI] [PubMed] [Google Scholar]
  9. Karlsson E., Eaker D., Ponterius G. Modification of amino groups in Naja naja neurotoxins and the preparation of radioactive derivatives. Biochim Biophys Acta. 1972 Feb 29;257(2):235–248. doi: 10.1016/0005-2795(72)90275-9. [DOI] [PubMed] [Google Scholar]
  10. Leprince P., Noble R. L., Hess G. P. Comparison of the interactions of a specific neurotoxin (alpha-bungarotoxin) with the acetylcholine receptor in Torpedo californica and Electrophorus electricus membrane preparations. Biochemistry. 1981 Sep 15;20(19):5565–5570. doi: 10.1021/bi00522a033. [DOI] [PubMed] [Google Scholar]
  11. Lo M. M., Garland P. B., Lamprecht J., Barnard E. A. Rotational mobility of the membrane-bound acetylcholine receptor of Torpedo electric organ measured by phosphorescence depolarisation. FEBS Lett. 1980 Mar 10;111(2):407–412. doi: 10.1016/0014-5793(80)80838-6. [DOI] [PubMed] [Google Scholar]
  12. Lukas R. J., Morimoto H., Hanley M. R., Bennett E. L. Radiolabeled alpha-bungarotoxin derivatives: kinetic interaction with nicotinic acetylcholine receptors. Biochemistry. 1981 Dec 22;20(26):7373–7378. doi: 10.1021/bi00529a008. [DOI] [PubMed] [Google Scholar]
  13. Maelicke A., Fulpius B. W., Klett R. P., Reich E. Acetylcholine receptor. Responses to drug binding. J Biol Chem. 1977 Jul 25;252(14):4811–4830. [PubMed] [Google Scholar]
  14. Reed K., Vandlen R., Bode J., Duguid J., Raftery M. A. Characterization of acetylcholine receptor-rich and acetylcholinesterase-rich membrane particles from Torpedo californica electroplax. Arch Biochem Biophys. 1975 Mar;167(1):138–144. doi: 10.1016/0003-9861(75)90449-x. [DOI] [PubMed] [Google Scholar]
  15. Schmidt J., Raftery M. A. A simple assay for the study of solubilized acetylcholine receptors. Anal Biochem. 1973 Apr;52(2):349–354. doi: 10.1016/0003-2697(73)90036-5. [DOI] [PubMed] [Google Scholar]
  16. Tsetlin V. I., Karlsson E., Arseniev A. S., Utkin Y. N., Surin A. M., Pashkov V. S., Pluzhnikov K. A., Ivanov V. T., Bystrov V. F., Ovchinnikov Y. A. EPR and fluorescence study of interaction of Naja naja oxiana neurotoxin II and its derivatives with acetylcholine receptor protein from Torpedo marmorata. FEBS Lett. 1979 Oct 1;106(1):47–52. doi: 10.1016/0014-5793(79)80692-4. [DOI] [PubMed] [Google Scholar]
  17. Weiland G. A., Molinoff P. B. Quantitative analysis of drug-receptor interactions: I. Determination of kinetic and equilibrium properties. Life Sci. 1981 Jul 27;29(4):313–330. doi: 10.1016/0024-3205(81)90324-6. [DOI] [PubMed] [Google Scholar]
  18. Weiland G., Georgia B., Wee V. T., Chignell C. F., Taylor P. Ligand interactions with cholinergic receptor-enriched membranes from Torpedo: influence of agonist exposure on receptor properties. Mol Pharmacol. 1976 Nov;12(6):1091–1105. [PubMed] [Google Scholar]

Articles from Biophysical Journal are provided here courtesy of The Biophysical Society

RESOURCES