Abstract
During transmitter release, synaptic vesicle membrane is specifically inserted into the nerve terminal plasma membrane only at specialized sites or "active zones." In an attempt to obtain a membrane fraction enriched in active zones, we have utilized the electric organ of the marine ray. From this organ, a fraction enriched in nerve terminals (synaptosomes) was prepared by conventional means. These synaptosomes were bound to microscopic beads by an antiserum to purified electric organ synaptic vesicles (anti-SV). The success of this immunoadsorption procedure was demonstrated by increased specific activities of bead- bound nerve terminal cytoplasmic markers and decreased specific activities of markers for contaminating membranes. To obtain a presynaptic plasma membrane (PSPM) fraction, we lysed the bead-bound synaptosomes by hypoosmotic shock and sonication, resulting in complete release of cytoplasmic markers. When the synaptosomal fraction was surface-labeled with iodine before immunoadsorption, 10% of this label remained bead-bound after lysis, compared with 2% of the total protein, indicating an approximately fivefold enrichment of bead-bound plasma membrane. Concomitantly, the specific activity of bead-bound anti-SV increased approximately 30-fold, indicating an enrichment of plasma membrane which contained inserted synaptic vesicle components. This PSPM preparation is not simply synaptic vesicle membrane since two- dimensional electrophoresis revealed that the polypeptides of the surface-iodinated PSPM preparation include both vesicle and numerous nonvesicle components. Secondly, antiserum to the PSPM fraction is markedly different from anti-SV and binds to external, nonvesicle, nerve terminal components.
Full Text
The Full Text of this article is available as a PDF (1.8 MB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Carlson S. S., Kelly R. B. An antiserum specific for cholinergic synaptic vesicles from electric organ. J Cell Biol. 1980 Oct;87(1):98–103. doi: 10.1083/jcb.87.1.98. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Carlson S. S., Wagner J. A., Kelly R. B. Purification of synaptic vesicles from elasmobranch electric organ and the use of biophysical criteria to demonstrate purity. Biochemistry. 1978 Apr 4;17(7):1188–1199. doi: 10.1021/bi00600a009. [DOI] [PubMed] [Google Scholar]
- Cohen C. M., Kalish D. I., Jacobson B. S., Branton D. Membrane isolation on polylysine-coated beads. Plasma membrane from HeLa cells. J Cell Biol. 1977 Oct;75(1):119–134. doi: 10.1083/jcb.75.1.119. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- 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]
- 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]
- Gentry M. K., Olsson R. A. A simple, specific, radioisotopic assay for 5'-nucleotidase. Anal Biochem. 1975 Apr;64(2):624–627. doi: 10.1016/0003-2697(75)90478-9. [DOI] [PubMed] [Google Scholar]
- Hooper J. E., Carlson S. S., Kelly R. B. Antibodies to synaptic vesicles purified from Narcine electric organ bind a subclass of mammalian nerve terminals. J Cell Biol. 1980 Oct;87(1):104–113. doi: 10.1083/jcb.87.1.104. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Ito A., Palade G. E. Presence of NADPH-cytochrome P-450 reductase in rat liver Golgi membranes. Evidence obtained by immunoadsorption method. J Cell Biol. 1978 Nov;79(2 Pt 1):590–597. doi: 10.1083/jcb.79.2.590. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Klinman N. R., Pickard A. R., Sigal N. H., Gearhart P. J., Metcalf E. S., Pierce S. K. Assessing B cell diversification by antigen receptor and precursor cell analysis. Ann Immunol (Paris) 1976 Jun-Jul;127(3-4):489–502. [PubMed] [Google Scholar]
- Kloog Y., Michaelson D. M., Sokolovsky M. Identification of muscarinic receptors in the Torpedo electric organ. Evidence for their presynaptic localization. FEBS Lett. 1978 Nov 15;95(2):331–334. doi: 10.1016/0014-5793(78)81023-0. [DOI] [PubMed] [Google Scholar]
- Medzihradsky F., Nandhasri P. S., Idoyaga-Vargas V., Sellinger O. Z. A comparison of ATPase activity of the glial cell fraction and the neuronal perikaryal fraction isolated in bulk from rat cerebbral cortex. J Neurochem. 1971 Aug;18(8):1599–1603. doi: 10.1111/j.1471-4159.1971.tb00023.x. [DOI] [PubMed] [Google Scholar]
- 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]
- Miljanich G. P., Brasier A. R., Kelly R. B. Partial purification of active zones of presynaptic plasma membrane by immunoadsorption. Biophys J. 1982 Jan;37(1):137–138. doi: 10.1016/S0006-3495(82)84640-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sanes J. R., Carlson S. S., Von Wedel R. J., Kelly R. B. Antiserum specific for motor nerve terminals in skeletal muscle. Nature. 1979 Aug 2;280(5721):403–404. doi: 10.1038/280403a0. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- 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]
- 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]
- Stanley P. E., Williams S. G. Use of the liquid scintillation spectrometer for determining adenosine triphosphate by the luciferase enzyme. Anal Biochem. 1969 Jun;29(3):381–392. doi: 10.1016/0003-2697(69)90323-6. [DOI] [PubMed] [Google Scholar]
- Vacquier V. D. The isolation of intact cortical granules from sea urchin eggs: calcium lons trigger granule discharge. Dev Biol. 1975 Mar;43(1):62–74. doi: 10.1016/0012-1606(75)90131-1. [DOI] [PubMed] [Google Scholar]
- Viratelle O. M., Bernhard S. A. Major component of acetylcholinesterase in Torpedo electroplax is not basal lamina associated. Biochemistry. 1980 Oct 28;19(22):4999–5007. doi: 10.1021/bi00563a011. [DOI] [PubMed] [Google Scholar]
- von Wedel R. J., Carlson S. S., Kelly R. B. Transfer of synaptic vesicle antigens to the presynaptic plasma membrane during exocytosis. Proc Natl Acad Sci U S A. 1981 Feb;78(2):1014–1018. doi: 10.1073/pnas.78.2.1014. [DOI] [PMC free article] [PubMed] [Google Scholar]