Neurochemical Research

, Volume 4, Issue 2, pp 137–153 | Cite as

Isolation of a synaptic junction-enriched fraction from the forebrain of day-old chickens

Preparation and characterization of chick forebrain subcellular fractions
  • Joseph C. Webster
  • Jack D. Klingman
Original Articles

Abstract

Synaptosomal plasma membrane (SPM) and other subcellular fractions were isolated from the forebrain of 1-day-old chickens by a procedure based on that of Davis and Bloom (16) and Cotman and Taylor (13). The procedure involves the centrifugation through a discontinuous sucrose gradient of a crude synaptosomal-mitochondrial fraction which has been lysed and weighted with iodonitrotetrazolium. SPM isolated by this method contains only small amounts of lysosomal or mitochondrial membranes and is practically devoid of contaminating microsomal membranes, as estimated by enzyme marker assays. The purity of chick-brain SPM prepared by this method is compared to the purity of chickbrain fractions obtained by two other laboratories, using different methods (4, 59). The SPM were extracted with Triton X-100 and all fractions solubilized in sodium dodecyl sulfate (SDS). The delipidated proteins of all fractions were subjected to SDS-polyacrylamide electrophoresis on slab gels and stained for protein. A distinct difference was observed between the patterns given by the Triton-soluble and-insoluble fractions. Electron microscopy of the synaptic junction fraction showed numerous junctional complexes.

Keywords

Sodium Dodecyl Sulfate Mitochondrial Membrane Sodium Dodecyl Sulfate Subcellular Fraction Sucrose Gradient 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Abdel-Latif, A. A. 1966. A simple method for isolation of nerve-ending particles from rat brain. Biochim. Biophys. Acta 121:403–406.PubMedGoogle Scholar
  2. 2.
    Appel, S. H., andDay, E. D. 1976. Cellular and subcellular fractionation. Pages 34–59,in Siegel, G. J., Albers, R. W., Katzman, R., andAgranoff, B. W. (eds.), Basic Neurochemistry, 2nd ed., Little, Brown, Boston.Google Scholar
  3. 3.
    Autilio, L., Appel, S. H., Pettis, P., andGambetti, P. L. 1968. Biochemical studies of synapses in vitro. I. Protein synthesis. Biochemistry 7:2615–2622.PubMedGoogle Scholar
  4. 4.
    Babitch, J. A., Breithaupt, T. B., Tien-Cheng Chiu, Garadi, R., andHelseth, D. L. 1976. Preparation of chick brain synaptosomes and synaptosomal membranes. Biochim. Biophys. Acta 433:75–89.PubMedGoogle Scholar
  5. 5.
    Barberis, C., Durand, M., Delorme, P., andGayet, J. 1977. Subcellular distribution of cytochromec oxidase, monoamine oxidase and lactate dehydrogenase in the developing chick telencephalon. Brain Res. 122:485–501.PubMedGoogle Scholar
  6. 6.
    Bloom, F. E., andAghajanian, G. K. 1966. Cytochemistry of synapses: Selective staining for electron microscopy. Science 154:1575–1577.PubMedGoogle Scholar
  7. 7.
    Bloom, F. E., andAghajanian, G. K. 1968. Fine structural and cytochemical analysis of the staining of synaptic junctions with phosphotungstic acid. J. Ultrastruct. Res. 22:361–375.PubMedGoogle Scholar
  8. 8.
    Carton, H. C., andAppel, S. H. 1974. Biochemical studies of transneural degeneration: The effect of enucleation of the biochemical maturation of the chick optic tectum. Brain Res. 67:289–306.PubMedGoogle Scholar
  9. 9.
    Cotman, C. W. 1975. Isolation of synaptosomes and synaptic plasma membrane fractions. Pages 445–452,Fleischer, S., andPacker, L. (eds.), Methods in Enzymology. Vol. 31A, Academic Press, New York.Google Scholar
  10. 10.
    Cotman, C. W., Levy, W., Banker, G., andTaylor, D. 1971. An ultrastructural and chemical analysis of the effect of Triton X-100 on synaptic plasma membranes. Biochim. Biophys. Acta 249:406–418.PubMedGoogle Scholar
  11. 11.
    Cotman, C. W., Mahler, H. R., andHugli, T. E. 1968. Isolation and characterization of insoluble proteins of the synaptic plasma membrane. Arch. Biochem. Biophys. 126:821–837.PubMedGoogle Scholar
  12. 12.
    Cotman, C. W., andMatthews, D. A. 1971. Synaptic plasma membranes from rat brain synaptosomes: Isolation and partial characterization. Biochim. Biophys. Acta 249:380–394.PubMedGoogle Scholar
  13. 13.
    Cotman, C. W., andTaylor, D. 1972. Isolation and structural studies on synaptic complexes from rat brain. J. Cell Biol. 55:696–711.PubMedGoogle Scholar
  14. 14.
    Cuenod, M., andSchonbach, J. 1971. Synaptic proteins and axonal flow in the pigeon visual pathway. J. Neurochem. 18:809–816.PubMedGoogle Scholar
  15. 15.
    Davis, G. A., andBloom, F. E. 1973. Subcellular particles separated through a histochemical reaction. Anal. Biochem. 51:429–435.PubMedGoogle Scholar
  16. 16.
    Davis, G. A., andBloom, F. E. 1973. Isolation of synaptic junctional complexes from rat brain. Brain Res. 62:135–153.PubMedGoogle Scholar
  17. 17.
    Den, H., andKaufman, B. 1968. Ganglioside and glycoprotein glycosytransferases in synaptosomes. Fed. Proc. 27:346.Google Scholar
  18. 18.
    DeRobertis, E., Pellegrino de Iraldi, A., Rodriguez de Lores Arnaiz, G., andSalganicoff, L. 1962. Cholinergic and noncholinergic nerve endings in rat brain. I. J. Neurochem. 9:23–35.PubMedGoogle Scholar
  19. 19.
    DeRobertis, E., Azcurra, J. M., andFiszer, S. 1967. Ultrastructure and cholinergic binding capacity of junctional complexes isolated from rat brain. Brain Res. 5:45–56.Google Scholar
  20. 20.
    Duncan, H. M., andMackler, B. 1966. Electron transport systems of yeast. III. Preparation and properties of cytochrome oxidase. J. Biol. Chem. 241:1694–1697.Google Scholar
  21. 21.
    Ellman, G. L., Courtnew, K. D., Andres, V., Jr., andFeatherstone, R. M. 1961. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7:88–95.PubMedGoogle Scholar
  22. 22.
    Fairbanks, G., Steck, T. L., andWallach, D. F. H. 1971. Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane.Biochemistry 10:2606–2617.PubMedGoogle Scholar
  23. 23.
    Fiszer, S., andDeRobertis, E. 1967. Action of Triton X-100 on ultrastructure and membrane bound enzymes of isolated nerve endings from rat brain. Brain Res. 5:31–44.PubMedGoogle Scholar
  24. 24.
    Goodrum, J. F., Bosmann, H. B., andTanaka, R. 1976. Glycoprotein: Galactosyl transferase in synaptic junction complexes isolated from rat forebrain. Neurosci. Abstr. 2:602.Google Scholar
  25. 25.
    Gray, E. G., andWhittaker, V. P. 1962. The isolation of nerve endings from brain and electron-microscopic study of cell fragments derived by homogenization and centrifugation. J. Anat. 96:79–88.PubMedGoogle Scholar
  26. 26.
    Gurd, J. W., Jones, L. R., Mahler, H. R., andMoore, W. J. 1974. Isolation and partial characterization of rat brain synaptic plasma membranes. J. Neurochem. 22:281–290.PubMedGoogle Scholar
  27. 27.
    Iqbal, Z., andTalwar, G. P. 1971. Acetylcholinesterase in developing chick embryo brain. J. Neurochem. 18:1261–1267.PubMedGoogle Scholar
  28. 28.
    Johnson, M. K., andWhittaker, V. P. 1963. Lactate dehydrogenase as a cytoplasmic marker in brain. Biochem. J. 88:404–409.PubMedGoogle Scholar
  29. 29.
    Jones, D. G., andBrearley, R. F. 1972. Further studies on synaptic junctions. I. Ultrastructural features in intact rat cerebral cortex. Z. Zellforsch. Mikrosk. Anat. 125:415–431.PubMedGoogle Scholar
  30. 30.
    Jones, D. G., andBrearley, R. F. 1972. Further studies on synaptic junctions. II. A comparison of synaptic ultrastructure in fractionated and intact cerebral cortex. Z. Zellforsch. Mikrosk. Anat. 125:432–447.PubMedGoogle Scholar
  31. 31.
    Jones, D. H., andMatus, A. I. 1974. Isolation of synaptic plasma membrane from brain by combined flotation-sedimentation density gradient centrifugation. Biochim. Biophys. Acta 356:276–287.PubMedGoogle Scholar
  32. 32.
    Kelly, P. T., andCotman, C. W. 1976. Intermolecular disulfide bonds at central nervous system synaptic junctions. Biochem. Biophys. Res. Commun. 73:858–864.PubMedGoogle Scholar
  33. 33.
    Kelly, P. T., andCotman, C. W. 1977. Identification of glycoproteins and proteins at synapses in the central nervous system. J. Biol. Chem. 252:786–793.PubMedGoogle Scholar
  34. 34.
    Klingman, G. I., Klingman, J. D., andPoliszczuk, A. 1968. Acetyl and pseudocholinesterase activities in sympathetic ganglia of rats. J. Neurochem. 15:1121–1130.PubMedGoogle Scholar
  35. 35.
    Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685.PubMedGoogle Scholar
  36. 36.
    Lagercrantz, H., andPertoft, H. 1972. Separation of catecholamine storing synaptosomes in colloidal silica density gradients. J. Neurochem. 19:811–823.PubMedGoogle Scholar
  37. 37.
    Levitan, I. B., Mushynski, W. E., andRamirez, G. 1972. Highly purified synaptosomal membranes from rat brain. Preparation and characterization. J. Biol. Chem. 247:5376–5381.PubMedGoogle Scholar
  38. 38.
    Livett, B. G., Rostas, J. A. P., Jeffrey, P. L., andAustin, L. 1974. Antigenicity of isolated synaptosomal membranes. Exp. Neurol. 43:330–338.PubMedGoogle Scholar
  39. 39.
    Lowry, O. H., Rosebrough, N. G., Farr, A. L., andRandall, R. J. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265–275.PubMedGoogle Scholar
  40. 40.
    Mahler, H. R., andCotman, C. W. 1970. Insoluble proteins of the synaptic plasma membrane. Pages 151–184,in Lajtha, A. Protein Metabolism of the Nervous System, Plenum Press, New York.Google Scholar
  41. 41.
    Margolis, R. K., Margolis, R. U., Preti, C., andLai, D. 1975. Distribution and metabolism of glycoproteins and glycosaminoglycans in subcellular fractions of brain. Biochemistry 14:4797–4808.PubMedGoogle Scholar
  42. 42.
    McBride, W. J., Aprison, H. M., andHingtgen, G. N. 1973. Effects of alpha-methyl-meta-tyrosine, alpha-methyl-meta-tyramine and metaraminol on the serotonin content in preparations of whole tissue and synaptosomes from the telencephalon of the pigeon. Neuropharmacology 12:769–776.PubMedGoogle Scholar
  43. 43.
    McBride, W. J., Mahler, H. R., Moore, W. J., andWhite, F. P. 1970. Isolation and characterization of membranes from rat cerebral cortex. J. Neurobiol. 2:73–92.PubMedGoogle Scholar
  44. 44.
    McBride, W. J., Perkins, J. G., Mahler, H. R., andMoore, W. J. 1972. Isolation of synaptosomes and synaptic membranes from the cerebral cortex of the rat, monkey, cat and pig. Life Sci. 11:975–987.Google Scholar
  45. 45.
    McBride, W. J., andVanTassel, J. 1972. Resolution of proteins from subfractions of nerve endings. Brain Res. 44:177–187.PubMedGoogle Scholar
  46. 46.
    Morgan, I. G., Wolfe, L. S., Mandel, P., andGombos, G. 1971. Isolation of plasma membranes from rat brain. Biochim. Biophys. Acta 241:737–751.PubMedGoogle Scholar
  47. 47.
    Nickerson, P. A. 1971. Fine structure of the Mongolian gerbil adrenal cortex. Anat. Rec. 171:443–456.PubMedGoogle Scholar
  48. 48.
    Oestreicher, A. B., andVanLeeuwen, C. 1975. Isolation and partial characterization of fractions enriched in synaptosomes from chick brain. J. Neurochem. 24:251–259.PubMedGoogle Scholar
  49. 49.
    Panzica Viglietti, C., Panzica, G. C., andGremo, F. 1977. Nerve endings isolated from chick embryonic optic tectum. I. Developmental aspects of intact synaptosomes. Experientia 33:458–460.PubMedGoogle Scholar
  50. 50.
    Pfenninger, K. H. 1971. The cytochemistry of synaptic densities. II. Proteinaceous components and mechanism of synaptic connectivity. J. Ultrastruct. Res. 34:451–475.Google Scholar
  51. 51.
    Rodriguez de Lores Arnaiz, G., Alberici, M., andDeRobertis, E. 1967. Ultrastructural and enzymatic studies of cholinergic and non-cholinergic synaptic membranes isolated from brain cortex. J. Neurochem. 14:215–225.PubMedGoogle Scholar
  52. 52.
    Rostas, J. A. P., Cotman, C. W., andKelly, P. T. 1977. The identification of membrane glycocomponents in polyacrylamide gels: A rapid method using125I-labelled lectins. Anal. Biochem. 80:366–372.PubMedGoogle Scholar
  53. 53.
    Sottocasa, G. L., Kuylenstierna, B., Ernster, L., andBergstrand, A. 1967. An electron-transport system associated with the outer membrane of liver mitochondria. A biochemical and morphological study. J. Cell Biol. 32:415–438.PubMedGoogle Scholar
  54. 54.
    Tamir, H., Rapport, M. M., andRoizin, L. 1974. Preparation of synaptosomes and vessicles with sodium diatrizoate. J. Neurochem. 23:943–949.PubMedGoogle Scholar
  55. 55.
    Tamir, H., Mahadik, S. P., andRapport, M. M. 1976. Fractionation of synaptic membranes with sodium diatrizoate. Anal. Biochem. 76:634–647.PubMedGoogle Scholar
  56. 56.
    Tettamanti, G., Bonali, F., Marchesini, S., andZambotti, V. 1973. A new procedure for the extraction, purification and fractionation of brain gangliosides. Biochim. Biophys. Acta 296:160–170.PubMedGoogle Scholar
  57. 57.
    Therien, H. M., andMushynski, W. E. 1976. Isolation of synaptic junctional complexes of high structural integrity from rat brain. J. Cell Biol. 71:807–822.PubMedGoogle Scholar
  58. 58.
    Tkachenko, A. N. 1972. Isolation of synaptosomal membrane from animal brains. Biokhimiya 37:248–254 (English translation, pp. 201–206).Google Scholar
  59. 59.
    Van Leeuwen, C., Stam, H., andOestreicher, A. B. 1976. Isolation and partial characterization of chick brain synaptic plasm membranes. Biochim. Biophys. Acta 436:53–67.PubMedGoogle Scholar
  60. 60.
    Whittaker, V. P. 1966. Some properties of synaptic membranes isolated from the central nervous system. Ann. N.Y. Acad. Sci. 137:982–998.PubMedGoogle Scholar
  61. 61.
    Whittaker, V. P., Michaelson, I. A., andKirkland, R. J. A. 1964. The separation of synaptic vessicles from nerve ending particles ('lssynaptosomes’). Biochem. J. 90:293–303.PubMedGoogle Scholar

Copyright information

© Plenum Publishing Corporation 1979

Authors and Affiliations

  • Joseph C. Webster
    • 1
  • Jack D. Klingman
    • 1
  1. 1.Department of BiochemistryState University of New York at BuffaloBuffalo

Personalised recommendations