Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1982 May 1;93(2):349–356. doi: 10.1083/jcb.93.2.349

Isolation of a presynaptic plasma membrane fraction from Torpedo cholinergic synaptosomes: evidence for a specific protein

PMCID: PMC2112834  PMID: 7096443

Abstract

Synaptosomal plasma membranes were isolated from Torpedo cholinergic synaptosomes which had been purified as previously described or repurified by equilibrium centrifugation. The synaptosomal plasma membrane could be distinguished from postsynaptic membranes by the absence of postsynaptic specific markers (nicotinic AChR) and by its low intramembrane particle complement after freeze fracture. In addition, the presynaptic membrane fraction contained acetylcholinesterase. Gel electrophoresis permitted the identification of a major protein component of the presynaptic membrane fraction which had a molecular weight of 67,000. This protein was not found in postsynaptic membrane or synaptic vesicle fractions. Thus it appeared to be specific to the nerve terminal plasma membrane.

Full Text

The Full Text of this article is available as a PDF (1.0 MB).

Selected References

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

  1. AMES B. N., DUBIN D. T. The role of polyamines in the neutralization of bacteriophage deoxyribonucleic acid. J Biol Chem. 1960 Mar;235:769–775. [PubMed] [Google Scholar]
  2. Allen T., Baerwald R., Potter L. T. Postsynaptic membranes in the electric tissue of Narcine: II. A freeze-fracture study of nicotinic receptor molecules. Tissue Cell. 1977;9(4):595–608. doi: 10.1016/0040-8166(77)90029-5. [DOI] [PubMed] [Google Scholar]
  3. Breer H., Morris S. J., Whittaker V. P. Adenosine triphosphatase activity associated with purified cholinergic synaptic vesicles of Torpedo marmorata. Eur J Biochem. 1977 Oct 17;80(1):313–318. doi: 10.1111/j.1432-1033.1977.tb11884.x. [DOI] [PubMed] [Google Scholar]
  4. Burger R. M., Lowenstein J. M. Preparation and properties of 5'-nucleotidase from smooth muscle of small intestine. J Biol Chem. 1970 Dec 10;245(23):6274–6280. [PubMed] [Google Scholar]
  5. Cartaud J., Benedetti E. L. A morphological study of the cholinergic receptor protein from Torpedo marmorata in its membrane environment and in its detergent-extracted purified form. J Cell Sci. 1978 Feb;29:313–337. doi: 10.1242/jcs.29.1.313. [DOI] [PubMed] [Google Scholar]
  6. Cohen J. B., Weber M., Huchet M., Changeux J. P. Purification from Torpedo marmorata electric tissue of membrane fragments particularly rich in cholinergic receptor protein. FEBS Lett. 1972 Oct 1;26(1):43–47. doi: 10.1016/0014-5793(72)80538-6. [DOI] [PubMed] [Google Scholar]
  7. Couteaux R., Pécot-Dechavassine M. Les zones spécialisées des membranes présynaptiques. C R Acad Sci Hebd Seances Acad Sci D. 1974 Jan 7;278(2):291–293. [PubMed] [Google Scholar]
  8. DE ROBERTIS E., PELLEGRINO DE IRALDI A., RODRIGUEZ G., GOMEZ C. J. On the isolation of nerve endings and synaptic vesicles. J Biophys Biochem Cytol. 1961 Jan;9:229–235. doi: 10.1083/jcb.9.1.229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dreyer F., Peper K., Akert K., Sandri C., Moor H. Ultrastructure of the "active zone" in the frog neuromuscular junction. Brain Res. 1973 Nov 23;62(2):373–380. doi: 10.1016/0006-8993(73)90699-9. [DOI] [PubMed] [Google Scholar]
  10. Duguid J. R., Raftery M. A. Fractionation and partial characterization of membrane particles from Torpedo californica electroplax. Biochemistry. 1973 Sep 11;12(19):3593–3597. doi: 10.1021/bi00743a003. [DOI] [PubMed] [Google Scholar]
  11. ELLMAN G. L., COURTNEY K. D., ANDRES V., Jr, FEATHER-STONE R. M. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol. 1961 Jul;7:88–95. doi: 10.1016/0006-2952(61)90145-9. [DOI] [PubMed] [Google Scholar]
  12. Feldberg W., Fessard A. The cholinergic nature of the nerves to the electric organ of the Torpedo (Torpedo marmorata). J Physiol. 1942 Aug 18;101(2):200–216. doi: 10.1113/jphysiol.1942.sp003975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fonnum F. A rapid radiochemical method for the determination of choline acetyltransferase. J Neurochem. 1975 Feb;24(2):407–409. doi: 10.1111/j.1471-4159.1975.tb11895.x. [DOI] [PubMed] [Google Scholar]
  14. GRAY E. G., WHITTAKER V. P. The isolation of nerve endings from brain: an electron-microscopic study of cell fragments derived by homogenization and centrifugation. J Anat. 1962 Jan;96:79–88. [PMC free article] [PubMed] [Google Scholar]
  15. Gulik-Krzywicki T., Costello M. J. The use of low temperature X-ray diffraction to evaluate freezing methods used in freeze-fracture electron microscopy. J Microsc. 1978 Jan;112(1):103–113. doi: 10.1111/j.1365-2818.1978.tb01158.x. [DOI] [PubMed] [Google Scholar]
  16. Heuser J. E., Reese T. S., Landis D. M. Functional changes in frog neuromuscular junctions studied with freeze-fracture. J Neurocytol. 1974 Mar;3(1):109–131. doi: 10.1007/BF01111936. [DOI] [PubMed] [Google Scholar]
  17. Israël M., Lesbats B. Continuous determination by a chemiluminescent method of acetylcholine release and compartmentation in Torpedo electric organ synaptosomes. J Neurochem. 1981 Dec;37(6):1475–1483. doi: 10.1111/j.1471-4159.1981.tb06317.x. [DOI] [PubMed] [Google Scholar]
  18. Israël M., Lesbats B. Détection continue de la libération d'acétylcholine de l'organe électrique de la Torpille à l'aide l'une réaction de chimiluminescence. C R Seances Acad Sci D. 1980 Oct 27;291(8):713–716. [PubMed] [Google Scholar]
  19. Israël M., Manaranche R., Marsal J., Meunier F. M., Morel N., Frachon P., Lesbats B. ATP-dependent calcium uptake by cholinergic synaptic vesicles isolated from Torpedo electric organ. J Membr Biol. 1980 May 23;54(2):115–126. doi: 10.1007/BF01940565. [DOI] [PubMed] [Google Scholar]
  20. Israël M., Manaranche R., Mastour-Frachon P., Morel N. Isolation of pure cholinergic nerve endings from the electric organ of Torpedo marmorata. Biochem J. 1976 Oct 15;160(1):113–115. doi: 10.1042/bj1600113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Israël M., Meunier F. M. The release of ATP triggered by transmitter action and its possible physiological significance: retrograde transmission. J Physiol (Paris) 1978;74(5):485–490. [PubMed] [Google Scholar]
  22. JOHNSON M. K., WHITTAKER V. P. LACTATE DEHYDROGENASE AS A CYTOPLASMIC MARKER IN BRAIN. Biochem J. 1963 Sep;88:404–409. doi: 10.1042/bj0880404. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kelly R. B., Deutsch J. W., Carlson S. S., Wagner J. A. Biochemistry of neurotransmitter release. Annu Rev Neurosci. 1979;2:399–446. doi: 10.1146/annurev.ne.02.030179.002151. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Lewis P. R., Shute C. C. An electron-microscopic study of cholinesterase distribution in the rat adrenal medulla. J Microsc. 1969;89(2):181–193. doi: 10.1111/j.1365-2818.1969.tb00664.x. [DOI] [PubMed] [Google Scholar]
  26. Meunier F. M., Morel N. Adenosine uptake by cholinergic synaptosomes from Torpedo electric organ. J Neurochem. 1978 Oct;31(4):845–851. doi: 10.1111/j.1471-4159.1978.tb00119.x. [DOI] [PubMed] [Google Scholar]
  27. Michaelson D. M., Ophir I. Sidedness of (calcium, magnesium) adenosine triphosphatase of purified Torpedo synaptic vesicles. J Neurochem. 1980 Jun;34(6):1483–1490. doi: 10.1111/j.1471-4159.1980.tb11228.x. [DOI] [PubMed] [Google Scholar]
  28. Michaelson D. M., Sokolovsky M. Induced acetylcholine release from active purely cholinergic Torpedo synaptosomes. J Neurochem. 1978 Jan;30(1):217–230. doi: 10.1111/j.1471-4159.1978.tb07055.x. [DOI] [PubMed] [Google Scholar]
  29. Morel N., Israel M., Manaranche R., Mastour-Frachon P. Isolation of pure cholinergic nerve endings from Torpedo electric organ. Evaluation of their metabolic properties. J Cell Biol. 1977 Oct;75(1):43–55. doi: 10.1083/jcb.75.1.43. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Morel N., Israël M., Manaranche R., Lesbats B. Stimulation of cholinergic synaptosomes isolated from Torpedo electric organ. Prog Brain Res. 1979;49:191–202. doi: 10.1016/s0079-6123(08)64633-6. [DOI] [PubMed] [Google Scholar]
  31. Morel N., Manaranche R., Gulik-Krzywicki T., Israel M. Ultrastructural changes and transmitter release induced by depolarization of cholinergic synaptosomes. A freeze-fracture study of a synaptosomal fraction from torpedo electric organ. J Ultrastruct Res. 1980 Mar;70(3):347–362. doi: 10.1016/s0022-5320(80)80017-7. [DOI] [PubMed] [Google Scholar]
  32. Morel N., Meunier F. M. Simultaneous release of acetylcholine and ATP from stimulated cholinergic synaptosomes. J Neurochem. 1981 May;36(5):1766–1773. doi: 10.1111/j.1471-4159.1981.tb00429.x. [DOI] [PubMed] [Google Scholar]
  33. Pfenninger K., Akert K., Moor H., Sandri C. The fine structure of freeze-fractured presynaptic membranes. J Neurocytol. 1972 Sep;1(2):129–149. doi: 10.1007/BF01099180. [DOI] [PubMed] [Google Scholar]
  34. Reale E., Luciano L., Spitznas M. The fine structural localization of acetylcholinesterase activity in the retina and optic nerve of rabbits. J Histochem Cytochem. 1971 Feb;19(2):85–96. doi: 10.1177/19.2.85. [DOI] [PubMed] [Google Scholar]
  35. Rosenbluth J. Synaptic membrane structure in Torpedo electric organ. J Neurocytol. 1975 Dec;4(6):697–712. doi: 10.1007/BF01181631. [DOI] [PubMed] [Google Scholar]
  36. Schaffner W., Weissmann C. A rapid, sensitive, and specific method for the determination of protein in dilute solution. Anal Biochem. 1973 Dec;56(2):502–514. doi: 10.1016/0003-2697(73)90217-0. [DOI] [PubMed] [Google Scholar]
  37. 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]
  38. Smith A. P., Loh H. H. Architecture of the nerve ending membrane. Life Sci. 1979 Jan 1;24(1):1–20. doi: 10.1016/0024-3205(79)90582-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. 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]
  40. Stadler H., Tashiro T. Isolation of synaptosomal plasma membranes from cholinergic nerve terminals and a comparison of their proteins with those of synaptic vesicles. Eur J Biochem. 1979 Nov 1;101(1):171–178. doi: 10.1111/j.1432-1033.1979.tb04229.x. [DOI] [PubMed] [Google Scholar]
  41. WHITTAKER V. P. The isolation and characterization of acetylcholine-containing particles from brain. Biochem J. 1959 Aug;72:694–706. doi: 10.1042/bj0720694. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Wagner J. A., Kelly R. B. Topological organization of proteins in an intracellular secretory organelle: the synaptic vesicle. Proc Natl Acad Sci U S A. 1979 Aug;76(8):4126–4130. doi: 10.1073/pnas.76.8.4126. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES