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. 1981 Oct 1;91(1):257–269. doi: 10.1083/jcb.91.1.257

Identification of a synaptic vesicle-specific membrane protein with a wide distribution in neuronal and neurosecretory tissue

PMCID: PMC2111938  PMID: 7298720

Abstract

Two different monoclonal antibodies, characterized initially as binding synaptic terminal regions of rat brain, bind a 65,000-dalton protein, which is exposed on the outer surface of brain synaptic vesicles. Immunocytochemical experiments at the electron microscope level demonstrate that these antibodies bind the vesicles in many different types of nerve terminals. The antibodies have been used successfully to purify synaptic vesicles from crude brain homogenates by immunoprecipitation onto the surface of polyacrylamide beads. The profiles of the structures precipitated by these beads are almost exclusively vesicular, confirming the vesicle-specificity of the antibodies. In SDS gels, the antibodies bind a single protein of 65,000 daltons. The two antibodies are not identical, but compete for binding sites on this protein. Immune competition experiments also demonstrate that the antigenic components on the 65,000-dalton protein are widely distributed in neuronal and neural secretory tissues. Detectable antigen is not found in uninnervated tissue--blood cells and extrajunctional muscle. Low levels are found in nonneural secretory tissues; it is not certain whether this reflects the presence of low amounts of the antigen on all the exocytotic vesicles in these tissues or whether the antigen is found only in neuronal fibers within these tissues. The molecular weight and at least two antigenic determinants of the 65,000-dalton protein are highly conserved throughout vertebrate phylogeny. The two antibodies recognize a 65,000-dalton protein present in shark, amphibia, birds, and mammals. The highly conserved nature of the determinants on this protein and their specific localization on secretory vesicles of many different types suggest that this protein may be essential for the normal function of neuronal secretory vesicles.

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Selected References

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  1. Babitch J. A., Benavides L. A. Comparison of synaptic plasma membrane and synaptic vesicle polypeptides by two-dimensional polyacrylamide gel electrophoresis. Neuroscience. 1979;4(5):603–613. doi: 10.1016/0306-4522(79)90137-4. [DOI] [PubMed] [Google Scholar]
  2. Bartlett S. F., Lagercrantz H., Smith A. D. Gel electrophoresis of soluble and insoluble proteins of noradrenergic vesicles from ox splenic nerve: a comparison with proteins of adrenal chromaffin granules. Neuroscience. 1976 Aug;1(4):339–344. doi: 10.1016/0306-4522(76)90061-0. [DOI] [PubMed] [Google Scholar]
  3. Bloom F. E., Ueda T., Battenberg E., Greengard P. Immunocytochemical localization, in synapses, of protein I, an endogenous substrate for protein kinases in mammalian brain. Proc Natl Acad Sci U S A. 1979 Nov;76(11):5982–5986. doi: 10.1073/pnas.76.11.5982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bretscher M. S., Thomson J. N., Pearse B. M. Coated pits act as molecular filters. Proc Natl Acad Sci U S A. 1980 Jul;77(7):4156–4159. doi: 10.1073/pnas.77.7.4156. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Burridge K. Direct identification of specific glycoproteins and antigens in sodium dodecyl sulfate gels. Methods Enzymol. 1978;50:54–64. doi: 10.1016/0076-6879(78)50007-4. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Castle J. D., Jamieson J. D., Palade G. E. Secretion granules of the rabbit parotid gland. Isolation, subfractionation, and characterization of the membrane and content subfractions. J Cell Biol. 1975 Jan;64(1):182–210. doi: 10.1083/jcb.64.1.182. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Castle J. D., Palade G. E. Secretion granules of the rabbit parotid. Selective removal of secretory contaminants from granule membranes. J Cell Biol. 1978 Feb;76(2):323–340. doi: 10.1083/jcb.76.2.323. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Da Prada M., Von Berlepsch K., Pletscher A. Storage of biogenic amines in blood platelets and adrenal medulla. Lack of evidence for direct involvement of glycosaminoglycans. Naunyn Schmiedebergs Arch Pharmacol. 1972;275(3):315–322. doi: 10.1007/BF00500059. [DOI] [PubMed] [Google Scholar]
  10. De Camilli P., Ueda T., Bloom F. E., Battenberg E., Greengard P. Widespread distribution of protein I in the central and peripheral nervous systems. Proc Natl Acad Sci U S A. 1979 Nov;76(11):5977–5981. doi: 10.1073/pnas.76.11.5977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fries E., Rothman J. E. Transport of vesicular stomatitis virus glycoprotein in a cell-free extract. Proc Natl Acad Sci U S A. 1980 Jul;77(7):3870–3874. doi: 10.1073/pnas.77.7.3870. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Goldstein J. L., Anderson R. G., Brown M. S. Coated pits, coated vesicles, and receptor-mediated endocytosis. Nature. 1979 Jun 21;279(5715):679–685. doi: 10.1038/279679a0. [DOI] [PubMed] [Google Scholar]
  13. Goodfellow P. N., Levinson J. R., Williams V. E., 2nd, McDevitt H. O. Monoclonal antibodies reacting with murine teratocarcinoma cells. Proc Natl Acad Sci U S A. 1979 Jan;76(1):377–380. doi: 10.1073/pnas.76.1.377. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. Hildebrand J. G., Barker D. L., Herbert E., Kravitz E. A. Screening for neurotransmitters: a rapid radiochemical procedure. J Neurobiol. 1971;2(3):231–246. doi: 10.1002/neu.480020305. [DOI] [PubMed] [Google Scholar]
  16. Hogue-Angeletti R. A., Sheetz P. B. A soluble lipid.protein complex from bovine adrenal medulla chromaffin granules. J Biol Chem. 1978 Aug 25;253(16):5613–5616. [PubMed] [Google Scholar]
  17. 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]
  18. Hope D. B., Pickup J. C. The isolation of neurosecretory granules from the posterior pituitary. Methods Enzymol. 1974;31:403–410. doi: 10.1016/0076-6879(74)31045-2. [DOI] [PubMed] [Google Scholar]
  19. Huber E., König P., Schuler G., Aberer W., Plattner H., Winkler H. Characterization and topography of the glycoproteins of adrenal chromaffin granules. J Neurochem. 1979 Jan;32(1):35–47. doi: 10.1111/j.1471-4159.1979.tb04507.x. [DOI] [PubMed] [Google Scholar]
  20. Hökfelt T., Johansson O., Ljungdahl A., Lundberg J. M., Schultzberg M. Peptidergic neurones. Nature. 1980 Apr 10;284(5756):515–521. doi: 10.1038/284515a0. [DOI] [PubMed] [Google Scholar]
  21. Inman J. K. Covalent linkage of functional groups, ligands, and proteins to polyacrylamide beads. Methods Enzymol. 1974;34:30–58. doi: 10.1016/s0076-6879(74)34006-2. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Ito M. Recent advances in cerebellar physiology and pathology. Adv Neurol. 1978;21:59–84. [PubMed] [Google Scholar]
  24. Jockusch B. M., Burger M. M., DaPrada M., Richards J. G., Chaponnier C., Gabbiani G. alpha-Actinin attached to membranes of secretory vesicles. Nature. 1977 Dec 15;270(5638):628–629. doi: 10.1038/270628a0. [DOI] [PubMed] [Google Scholar]
  25. Jones D. H., Matus A. I. Isolation of synaptic plasma membrane from brain by combined flotation-sedimentation density gradient centrifugation. Biochim Biophys Acta. 1974 Aug 9;356(3):276–287. doi: 10.1016/0005-2736(74)90268-5. [DOI] [PubMed] [Google Scholar]
  26. Kanner B. I., Sharon I., Maron R., Schuldiner S. Electrogenic transport of biogenic amines in chromaffin granule membrane vesicles. FEBS Lett. 1980 Feb 25;111(1):83–86. doi: 10.1016/0014-5793(80)80766-6. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. Klinman N. R. The mechanism of antigenic stimulation of primary and secondary clonal precursor cells. J Exp Med. 1972 Aug 1;136(2):241–260. doi: 10.1084/jem.136.2.241. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. Lagercrantz H. On the composition and function of large dense cored vesicles in sympathetic nerves. Neuroscience. 1976;1(2):81–92. doi: 10.1016/0306-4522(76)90002-6. [DOI] [PubMed] [Google Scholar]
  31. Meyer D. I., Burger M. M. The chromaffin granule surface: the presence of actin and the nature of its interaction with the membrane. FEBS Lett. 1979 May 1;101(1):129–133. doi: 10.1016/0014-5793(79)81310-1. [DOI] [PubMed] [Google Scholar]
  32. Moore R. Y., Bloom F. E. Central catecholamine neuron systems: anatomy and physiology of the norepinephrine and epinephrine systems. Annu Rev Neurosci. 1979;2:113–168. doi: 10.1146/annurev.ne.02.030179.000553. [DOI] [PubMed] [Google Scholar]
  33. Palade G. Intracellular aspects of the process of protein synthesis. Science. 1975 Aug 1;189(4200):347–358. doi: 10.1126/science.1096303. [DOI] [PubMed] [Google Scholar]
  34. Parks D. R., Bryan V. M., Oi V. T., Herzenberg L. A. Antigen-specific identification and cloning of hybridomas with a fluorescence-activated cell sorter. Proc Natl Acad Sci U S A. 1979 Apr;76(4):1962–1966. doi: 10.1073/pnas.76.4.1962. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Patterson P. H., Reichardt L. F., Chun L. L. Biochemical studies on the development of primary sympathetic neurons in cell culture. Cold Spring Harb Symp Quant Biol. 1976;40:389–397. doi: 10.1101/sqb.1976.040.01.037. [DOI] [PubMed] [Google Scholar]
  36. Roda L. G., Hogue-Angeletti R. A. Peptides in the adrenal medulla chromaffin granule. FEBS Lett. 1979 Nov 15;107(2):393–397. doi: 10.1016/0014-5793(79)80415-9. [DOI] [PubMed] [Google Scholar]
  37. Schachner M., Hedley-Whyte E. T., Hsu D. W., Schoonmaker G., Bignami A. Ultrastructural localization of glial fibrillary acidic protein in mouse cerebellum by immunoperoxidase labeling. J Cell Biol. 1977 Oct;75(1):67–73. doi: 10.1083/jcb.75.1.67. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Swanson L. W., Hartman B. K. The central adrenergic system. An immunofluorescence study of the location of cell bodies and their efferent connections in the rat utilizing dopamine-beta-hydroxylase as a marker. J Comp Neurol. 1975 Oct 15;163(4):467–505. doi: 10.1002/cne.901630406. [DOI] [PubMed] [Google Scholar]
  39. Tartakoff A., Greene L. J., Palade G. E. Studies on the guinea pig pancreas. Fractionation and partial characterization of exocrine proteins. J Biol Chem. 1974 Dec 10;249(23):7420–7431. [PubMed] [Google Scholar]
  40. Uchizono K., Ohsawa K., Ikemoto H., Al-Samarrai S. Morphological and biochemical properties of synaptic vesicles isolated from guinea pig brain. Neuroscience. 1979;4(5):593–601. doi: 10.1016/0306-4522(79)90136-2. [DOI] [PubMed] [Google Scholar]
  41. Uvnäs B. The isolation of secretory granules from mast cells. Methods Enzymol. 1974;31:395–402. doi: 10.1016/0076-6879(74)31044-0. [DOI] [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]
  43. Wang Y. J., Mahler H. R. Topography of the synaptosomal membrane. J Cell Biol. 1976 Nov;71(2):639–658. doi: 10.1083/jcb.71.2.639. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Winkler H. The composition of adrenal chromaffin granules: an assessment of controversial results. Neuroscience. 1976;1(2):65–80. doi: 10.1016/0306-4522(76)90001-4. [DOI] [PubMed] [Google Scholar]
  45. Zisapel N., Levi M., Gozes I. Tubulin: an integral protein of mammalian synaptic vesicle membranes. J Neurochem. 1980 Jan;34(1):26–32. doi: 10.1111/j.1471-4159.1980.tb04617.x. [DOI] [PubMed] [Google Scholar]

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