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. 1972 Jul;128(4):833–845. doi: 10.1042/bj1280833

The isolation of pure cholinergic synaptic vesicles from the electric organs of elasmobranch fish of the family Torpedinidae

V P Whittaker 1, W B Essman 1,*, G H C Dowe 1
PMCID: PMC1173903  PMID: 4638794

Abstract

1. Zonal centrifuging permitted the separation, on the milligram scale and in a form largely free from contamination by soluble cytoplasmic protein or membrane fragments derived from other structures, of synaptic vesicles from the purely cholinergic terminals of the electric organ of Torpedo. Up to 100g of tissue could be processed in a single run. 2. As much as 46% of the bound acetylcholine from the original tissue preparation was recovered as a single peak of density equivalent to 0.38m-sucrose–0.21m-NaCl and with a concentration of up to 680nmol of acetylcholine/mg of protein. 3. The limiting concentration of acetylcholine in isolated vesicles when allowance had been made for non-vesicular protein appeared to be about 600nmol/mg of protein. 4. Vesicle counts by a `bead-tagging' procedure indicated an acetylcholine content of about 360μmol/ml of vesicles; thus the vesicle protein content would be about 60% (w/v). 5. Calculations showed that the core of the vesicle, accounting for about 55% of the vesicle volume, could be largely filled with acetylcholine and protein.

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Selected References

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

  1. ALDRIDGE W. N., EMERY R. C., STREET B. W. A tissue homogenizer. Biochem J. 1960 Nov;77:326–327. doi: 10.1042/bj0770326. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bennett M. V. Comparative physiology: electric organs. Annu Rev Physiol. 1970;32:471–528. doi: 10.1146/annurev.ph.32.030170.002351. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. FRONTALI N., TOSCHI G. Subcellular fractions from the electric tissue of Torpedo; morphological aspect and cholinesterase content. Exp Cell Res. 1958 Oct;15(2):446–450. doi: 10.1016/0014-4827(58)90055-7. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. Fonnum F. A radiochemical method for the estimation of choline acetyltransferase. Biochem J. 1966 Aug;100(2):479–484. doi: 10.1042/bj1000479. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Fonnum F. The 'compartmentation' of choline acetyltransferase within the synaptosome. Biochem J. 1967 Apr;103(1):262–270. doi: 10.1042/bj1030262. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. HEBB C. O., WAITES G. M. Choline acetylase in antero- and retro-grade degeneration of a cholinergic nerve. J Physiol. 1956 Jun 28;132(3):667–671. doi: 10.1113/jphysiol.1956.sp005556. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. HEBB C. O., WHITTAKER V. P. Intracellular distributions of acetylcholine and choline acetylase. J Physiol. 1958 Jun 18;142(1):187–196. doi: 10.1113/jphysiol.1958.sp006008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Israel M., Gautron J., Lesbats B. Isolement des vésicules synaptiques de l'organe électrique de la Torpille et localisation de l'acétylcholine à leur niveau. C R Acad Sci Hebd Seances Acad Sci D. 1968 Jan 15;266(3):273–275. [PubMed] [Google Scholar]
  12. Israël M., Gautron J., Lesbats B. Fractionnement de l'organe electrique de la torpille: localisation subcellulaire de l'acetylcholine. J Neurochem. 1970 Oct;17(10):1441–1450. doi: 10.1111/j.1471-4159.1970.tb00511.x. [DOI] [PubMed] [Google Scholar]
  13. JOHNSON M. K. The intracellular distribution of glycolytic and other enzymes in rat-brain homogenates and mitochondrial preparations. Biochem J. 1960 Dec;77:610–618. doi: 10.1042/bj0770610. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. LAVERTY R., MICHAELSON I. A., SHARMAN D. F., WHITTAKER V. P. THE SUBCELLULAR LOCALIZATION OF DOPAMINE AND ACETYLCHOLINE IN THE DOG CAUDATE NUCLEUS. Br J Pharmacol Chemother. 1963 Dec;21:482–490. doi: 10.1111/j.1476-5381.1963.tb02016.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  16. Soifer D., Whittaker V. P. Morphology of subcellular fractions derived from the electric organ of Torpedo. Biochem J. 1972 Jul;128(4):845–846. doi: 10.1042/bj1280845. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Trejo A. G., Haddock J. W., Chittenden G. J., Baddiley J. The biosynthesis of galactofuranosyl residues in galactocarolose. Biochem J. 1971 Mar;122(1):49–57. doi: 10.1042/bj1220049. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. WHITTAKER V. P., SHERIDAN M. N. THE MORPHOLOGY AND ACETYLCHOLINE CONTENT OF ISOLATED CEREBRAL CORTICAL SYNAPTIC VESICLES. J Neurochem. 1965 May;12:363–372. doi: 10.1111/j.1471-4159.1965.tb04237.x. [DOI] [PubMed] [Google Scholar]
  19. Whittaker V. P., Michaelson I. A., Kirkland R. J. The separation of synaptic vesicles from nerve-ending particles ('synaptosomes'). Biochem J. 1964 Feb;90(2):293–303. doi: 10.1042/bj0900293. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Whittaker V. P. Origin and function of synaptic vesicles. Ann N Y Acad Sci. 1971 Sep 15;183:21–32. doi: 10.1111/j.1749-6632.1971.tb30739.x. [DOI] [PubMed] [Google Scholar]
  21. Whittaker V. P. Subcellular localization of neurotransmitters. Adv Cytopharmacol. 1971 May;1:319–330. [PubMed] [Google Scholar]

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