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. 1984 Oct;3(10):2287–2290. doi: 10.1002/j.1460-2075.1984.tb02127.x

The 43-K protein, v1, associated with acetylcholine receptor containing membrane fragments is an actin-binding protein.

J H Walker, C M Boustead, V Witzemann
PMCID: PMC557681  PMID: 6389118

Abstract

Acetylcholine receptor enriched membrane fragments were obtained from the electric organs of Torpedo marmorata. The purified membrane fragments contained several proteins in addition to the acetylcholine receptor subunits. One of these was shown to be actin by means of immune blotting with a monoclonal antibody. Brief treatment of the membranes with pH 11.0 buffer removed actin and the other non-receptor proteins including the receptor-associated 43 000 mol. wt. polypeptide. This polypeptide was shown to bind actin after transferring the proteins from one- and two-dimensional polyacrylamide gels to nitrocellulose paper and incubating the nitrocellulose blots with actin. Specifically bound actin was demonstrated using the monoclonal antibodies to actin. No calcium or calmodulin dependency of binding was observed. The findings suggest that the 43 000 mol. wt. polypeptide is a link between the membrane-bound acetylcholine receptor and the cytoskeleton.

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Selected References

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

  1. Barrantes F. J., Neugebauer D. C., Zingsheim H. P. Peptide extraction by alkaline treatment is accompanied by rearrangement of the membrane-bound acetylcholine receptor from Torpedo marmorata. FEBS Lett. 1980 Mar 24;112(1):73–78. doi: 10.1016/0014-5793(80)80131-1. [DOI] [PubMed] [Google Scholar]
  2. Barrantes F. J. Oligomeric forms of the membrane-bound acetylcholine receptor disclosed upon extraction of the Mr 43,000 nonreceptor peptide. J Cell Biol. 1982 Jan;92(1):60–68. doi: 10.1083/jcb.92.1.60. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bartholdi M., Barrantes F. J., Jovin T. M. Rotational molecular dynamics of the membrane-bound acetylcholine receptor revealed by phosphorescence spectroscopy. Eur J Biochem. 1981 Nov;120(2):389–397. doi: 10.1111/j.1432-1033.1981.tb05716.x. [DOI] [PubMed] [Google Scholar]
  4. Brown S. S., Yamamoto K., Spudich J. A. A 40,000-dalton protein from Dictyostelium discoideum affects assembly properties of actin in a Ca2+-dependent manner. J Cell Biol. 1982 Apr;93(1):205–210. doi: 10.1083/jcb.93.1.205. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Burden S. J., DePalma R. L., Gottesman G. S. Crosslinking of proteins in acetylcholine receptor-rich membranes: association between the beta-subunit and the 43 kd subsynaptic protein. Cell. 1983 Dec;35(3 Pt 2):687–692. doi: 10.1016/0092-8674(83)90101-0. [DOI] [PubMed] [Google Scholar]
  6. Burden S. Identification of an intracellular postsynaptic antigen at the frog neuromuscular junction. J Cell Biol. 1982 Sep;94(3):521–530. doi: 10.1083/jcb.94.3.521. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cartaud J., Sobel A., Rousselet A., Devaux P. F., Changeux J. P. Consequences of alkaline treatment for the ultrastructure of the acetylcholine-receptor-rich membranes from Torpedo marmorata electric organ. J Cell Biol. 1981 Aug;90(2):418–426. doi: 10.1083/jcb.90.2.418. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Changeux J. P. The acetylcholine receptor: an "allosteric" membrane protein. Harvey Lect. 1979 1980;75:85–254. [PubMed] [Google Scholar]
  9. Condeelis J., Vahey M. A calcium- and pH-regulated protein from Dictyostelium discoideum that cross-links actin filaments. J Cell Biol. 1982 Aug;94(2):466–471. doi: 10.1083/jcb.94.2.466. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Elliott J., Blanchard S. G., Wu W., Miller J., Strader C. D., Hartig P., Moore H. P., Racs J., Raftery M. A. Purification of Torpedo californica post-synaptic membranes and fractionation of their constituent proteins. Biochem J. 1980 Mar 1;185(3):667–677. doi: 10.1042/bj1850667. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Elliott J., Dunn S. M., Blanchard S. G., Raftery M. A. Specific binding of perhydrohistrionicotoxin to Torpedo acetylcholine receptor. Proc Natl Acad Sci U S A. 1979 Jun;76(6):2576–2579. doi: 10.1073/pnas.76.6.2576. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fox G. Q., Richardson G. P. The developmental morphology of Torpedo marmorata: electric organ--electrogenic phase. J Comp Neurol. 1979 May 15;185(2):293–315. doi: 10.1002/cne.901850205. [DOI] [PubMed] [Google Scholar]
  14. Gysin R., Wirth M., Flanagan S. D. Structural heterogeneity and subcellular distribution of nicotinic synapse-associated proteins. J Biol Chem. 1981 Nov 25;256(22):11373–11376. [PubMed] [Google Scholar]
  15. Gysin R., Yost B., Flanagan S. D. Immunochemical and molecular differentiation of 43 000 molecular weight proteins associated with Torpedo neuroelectrocyte synapses. Biochemistry. 1983 Dec 6;22(25):5781–5789. doi: 10.1021/bi00294a016. [DOI] [PubMed] [Google Scholar]
  16. Hasegawa T., Takahashi S., Hayashi H., Hatano S. Fragmin: a calcium ion sensitive regulatory factor on the formation of actin filaments. Biochemistry. 1980 Jun 10;19(12):2677–2683. doi: 10.1021/bi00553a021. [DOI] [PubMed] [Google Scholar]
  17. Heuser J. E., Salpeter S. R. Organization of acetylcholine receptors in quick-frozen, deep-etched, and rotary-replicated Torpedo postsynaptic membrane. J Cell Biol. 1979 Jul;82(1):150–173. doi: 10.1083/jcb.82.1.150. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hinssen H. An actin-modulating protein from Physarum polycephalum. I. Isolation and purification. Eur J Cell Biol. 1981 Feb;23(2):225–233. [PubMed] [Google Scholar]
  19. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Neubig R. R., Krodel E. K., Boyd N. D., Cohen J. B. Acetylcholine and local anesthetic binding to Torpedo nicotinic postsynaptic membranes after removal of nonreceptor peptides. Proc Natl Acad Sci U S A. 1979 Feb;76(2):690–694. doi: 10.1073/pnas.76.2.690. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Nghiêm H. O., Cartaud J., Dubreuil C., Kordeli C., Buttin G., Changeux J. P. Production and characterization of a monoclonal antibody directed against the 43,000-dalton v1 polypeptide from Torpedo marmorata electric organ. Proc Natl Acad Sci U S A. 1983 Oct;80(20):6403–6407. doi: 10.1073/pnas.80.20.6403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. O'Farrell P. H. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975 May 25;250(10):4007–4021. [PMC free article] [PubMed] [Google Scholar]
  24. Porter S., Froehner S. C. Characterization and localization of the Mr = 43,000 proteins associated with acetylcholine receptor-rich membranes. J Biol Chem. 1983 Aug 25;258(16):10034–10040. [PubMed] [Google Scholar]
  25. Saitoh T., Wennogle L. P., Changeux J. P. Factors regulating the susceptibility of the acetylcholine receptor protein to heat inactivation. FEBS Lett. 1979 Dec 15;108(2):489–494. doi: 10.1016/0014-5793(79)80595-5. [DOI] [PubMed] [Google Scholar]
  26. Sealock R. Cytoplasmic surface structure in postsynaptic membranes from electric tissue visualized by tannic-acid-mediated negative contrasting. J Cell Biol. 1982 Feb;92(2):514–522. doi: 10.1083/jcb.92.2.514. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Snabes M. C., Boyd A. E., 3rd, Bryan J. Detection of actin-binding proteins in human platelets by 125I-actin overlay of polyacrylamide gels. J Cell Biol. 1981 Sep;90(3):809–812. doi: 10.1083/jcb.90.3.809. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Sobel A., Weber M., Changeux J. P. Large-scale purification of the acetylcholine-receptor protein in its membrane-bound and detergent-extracted forms from Torpedo marmorata electric organ. Eur J Biochem. 1977 Oct 17;80(1):215–224. doi: 10.1111/j.1432-1033.1977.tb11874.x. [DOI] [PubMed] [Google Scholar]
  29. St John P. A., Froehner S. C., Goodenough D. A., Cohen J. B. Nicotinic postsynaptic membranes from Torpedo: sidedness, permeability to macromolecules, and topography of major polypeptides. J Cell Biol. 1982 Feb;92(2):333–342. doi: 10.1083/jcb.92.2.333. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Strader C. D., Lazarides E., Raftery M. A. The characterization of actin associated with postsynaptic membranes from Torpedo californica. Biochem Biophys Res Commun. 1980 Jan 29;92(2):365–373. doi: 10.1016/0006-291x(80)90342-3. [DOI] [PubMed] [Google Scholar]
  31. Walker J. H., Obrocki J., Zimmermann C. W. Identification of a proteoglycan antigen characteristic of cholinergic synaptic vesicles. J Neurochem. 1983 Jul;41(1):209–216. doi: 10.1111/j.1471-4159.1983.tb11829.x. [DOI] [PubMed] [Google Scholar]
  32. Witzemann V., Schmid D., Boustead C. Differentiation-dependent changes of nicotinic synapse-associated proteins. Eur J Biochem. 1983 Mar 15;131(2):235–245. doi: 10.1111/j.1432-1033.1983.tb07255.x. [DOI] [PubMed] [Google Scholar]

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