Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1976 Dec 1;71(3):807–822. doi: 10.1083/jcb.71.3.807

Isolation of synaptic junctional complexes of high structural integrity from rat brain

PMCID: PMC2109788  PMID: 186464

Abstract

A new method has been developed for isolating synaptic junctional complexes (SJC) of high structural integrity. The major step in the isolation involves homogenization of a synaptosomal membrane (SM) fraction in a biphasic system consisting of Freon 113 and an aqueous phase containing 0.2% Triton X-100. Well-preserved SJCs, along with membrane vesicles, were recovered in the aqueous phase after low-speed centrifugation of the homogenate. The membranes were subsequently separated from the SJCs by centrifugation on a discontinuous sucrose density gradient. The purity and identity of subcellular fractions were monitored by thin sectioning electron microscopy, using specific and nonspecific staining methods. From the electron microscope studies we conclude that SJCs and their components occupy about 65% of the area covered by structures in this fraction. The assay of enzyme activities indicates that homogenization in Triton-Freon and subsequent steps of the isolation procedure affect the activities of Na, K-ATPase, cytochrome oxidase, and acid phosphatase to different extents, but do not cause total inactivation. Electrophoresis of the SJC-enriched fraction on sodium dodecyl sulfate-polyacrylamide gels has demonstrated that a polypeptide which co-migrates with tubulin is the major component in this fraction, and that a polypeptide co-migrating with actin is also present.

Full Text

The Full Text of this article is available as a PDF (3.5 MB).

Selected References

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

  1. Achee F. M., Togulga G., Gabay S. Studies of monoamine oxidases: properties of the enzyme in bovine and rabbit brain mitochondria. J Neurochem. 1974 May;22(5):651–661. doi: 10.1111/j.1471-4159.1974.tb04277.x. [DOI] [PubMed] [Google Scholar]
  2. Awasthi Y. C., Chuang T. F., Keenan T. W., Crane F. L. Tightly bound cardiolipin in cytochrome oxidase. Biochim Biophys Acta. 1971 Jan 12;226(1):42–52. doi: 10.1016/0005-2728(71)90176-9. [DOI] [PubMed] [Google Scholar]
  3. Banker G., Churchill L., Cotman C. W. Proteins of the postsynaptic density. J Cell Biol. 1974 Nov;63(2 Pt 1):456–465. doi: 10.1083/jcb.63.2.456. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Berl S., Puszkin S., Nicklas W. J. Actomyosin-like protein in brain. Science. 1973 Feb 2;179(4072):441–446. doi: 10.1126/science.179.4072.441. [DOI] [PubMed] [Google Scholar]
  5. Berlin R. D., Oliver J. M., Ukena T. E., Yin H. H. Control of cell surface topography. Nature. 1974 Jan 4;247(5435):45–46. doi: 10.1038/247045a0. [DOI] [PubMed] [Google Scholar]
  6. Blitz A. L., Fine R. E. Muscle-like contractile proteins and tubulin in synaptosomes. Proc Natl Acad Sci U S A. 1974 Nov;71(11):4472–4476. doi: 10.1073/pnas.71.11.4472. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bloom F. E., Aghajanian G. K. Cytochemistry of synapses: selective staining for electron microscopy. Science. 1966 Dec 23;154(3756):1575–1577. doi: 10.1126/science.154.3756.1575. [DOI] [PubMed] [Google Scholar]
  8. Bloom F. E., Aghajanian G. K. Fine structural and cytochemical analysis of the staining of synaptic junctions with phosphotungstic acid. J Ultrastruct Res. 1968 Mar;22(5):361–375. doi: 10.1016/s0022-5320(68)90027-0. [DOI] [PubMed] [Google Scholar]
  9. COLONNIER M., GUILLERY R. W. SYNAPTIC ORGANIZATION IN THE LATERAL GENICULATE NUCLEUS OF THE MONKEY. Z Zellforsch Mikrosk Anat. 1964 Apr 9;62:333–355. doi: 10.1007/BF00339284. [DOI] [PubMed] [Google Scholar]
  10. Cotman C. W., Banker G., Churchill L., Taylor D. Isolation of postsynaptic densities from rat brain. J Cell Biol. 1974 Nov;63(2 Pt 1):441–455. doi: 10.1083/jcb.63.2.441. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Cotman C. W., Banker G., Levy W., Taylor D. An ultrastructural and chemical analysis of the effect of triton X-100 on synaptic plasma membranes. Biochim Biophys Acta. 1971 Dec 3;249(2):406–418. doi: 10.1016/0005-2736(71)90119-2. [DOI] [PubMed] [Google Scholar]
  12. Cotman C. W., Matthews D. A. Synaptic plasma membranes from rat brain synaptosomes: isolation and partial characterization. Biochim Biophys Acta. 1971 Dec 3;249(2):380–394. doi: 10.1016/0005-2736(71)90117-9. [DOI] [PubMed] [Google Scholar]
  13. Cotman C. W., Taylor D. Isolation and structural studies on synaptic complexes from rat brain. J Cell Biol. 1972 Dec;55(3):696–711. doi: 10.1083/jcb.55.3.696. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Davis G. A., Bloom F. E. Isolation of synaptic junctional complexes from rat brain. Brain Res. 1973 Nov 9;62(1):135–153. doi: 10.1016/0006-8993(73)90624-0. [DOI] [PubMed] [Google Scholar]
  15. Davison P. F., Winslow B. The protein subunit of calf brain neurofilament. J Neurobiol. 1974;5(2):119–133. doi: 10.1002/neu.480050204. [DOI] [PubMed] [Google Scholar]
  16. DiPietro D. L., Zengerle F. S. Separation and properties of three acid phosphatases from human placenta. J Biol Chem. 1967 Jul 25;242(14):3391–3395. [PubMed] [Google Scholar]
  17. Duncan H. M., Mackler B. Electron transport systems of yeast. 3. Preparation and properties of cytochrome oxidase. J Biol Chem. 1966 Apr 25;241(8):1694–1697. [PubMed] [Google Scholar]
  18. Edelman G. M., Yahara I., Wang J. L. Receptor mobility and receptor-cytoplasmic interactions in lymphocytes. Proc Natl Acad Sci U S A. 1973 May;70(5):1442–1446. doi: 10.1073/pnas.70.5.1442. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Fiszer S., Robertis E. D. Action of triton X-100 on ultrastructure and membrane-bound- enzymes of isolated nerve endings from rat brain. Brain Res. 1967 May;5(1):31–44. doi: 10.1016/0006-8993(67)90217-x. [DOI] [PubMed] [Google Scholar]
  20. GRAY E. G. Axo-somatic and axo-dendritic synapses of the cerebral cortex: an electron microscope study. J Anat. 1959 Oct;93:420–433. [PMC free article] [PubMed] [Google Scholar]
  21. Gray E. G. Presynaptic microtubules and their association with synaptic vesicles. Proc R Soc Lond B Biol Sci. 1975 Aug 19;190(1100):367–372. [PubMed] [Google Scholar]
  22. Gray E. G. Synaptic fine structure and nuclear, cytoplasmic and extracellular networks: The stereoframework concept. J Neurocytol. 1975 Jun;4(3):315–339. doi: 10.1007/BF01102116. [DOI] [PubMed] [Google Scholar]
  23. Gray E. G. The cytonet, plain and coated vesicles, reticulosomes, multivesicular bodies and nuclear pores. Brain Res. 1973 Nov 23;62(2):329–335. doi: 10.1016/0006-8993(73)90693-8. [DOI] [PubMed] [Google Scholar]
  24. Helenius A., Simons K. Solubilization of membranes by detergents. Biochim Biophys Acta. 1975 Mar 25;415(1):29–79. doi: 10.1016/0304-4157(75)90016-7. [DOI] [PubMed] [Google Scholar]
  25. Hosie R. J. The localization of adenosine triphosphatases in morphologically characterized subcellular fractions of guinea-pig brain. Biochem J. 1965 Aug;96(2):404–412. doi: 10.1042/bj0960404. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. KURTZ S. M. A new method for embedding tissues in Vestopal W. J Ultrastruct Res. 1961 Oct;5:468–469. doi: 10.1016/s0022-5320(61)80020-8. [DOI] [PubMed] [Google Scholar]
  27. Kadota K., Kadota T. Letter: Isolation of coated vesicles, plain synaptic vesicles and fine particles from synaptosomes of guinea pig whole brain. J Electron Microsc (Tokyo) 1973;22(1):91–98. [PubMed] [Google Scholar]
  28. Kornguth S. E., Sunderland E. Isolation and partial characterization of a tubulin-like protein from human and swine synaptosomal membranes. Biochim Biophys Acta. 1975 May 30;393(1):100–114. doi: 10.1016/0005-2795(75)90220-2. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. Le Beux Y. J. An ultrastructural study of the synaptic densities, nematosomes, neurotubules, neurofilaments and of a further three-dimensional filamentous network as disclosed by the E-PTA staining procedure. Z Zellforsch Mikrosk Anat. 1973;143(2):239–272. doi: 10.1007/BF00307481. [DOI] [PubMed] [Google Scholar]
  31. Matus A. I., Walters B. B. Ultrastructure of the synaptic junctional lattice isolated from mammalian brain. J Neurocytol. 1975 Jun;4(3):369–375. doi: 10.1007/BF01102119. [DOI] [PubMed] [Google Scholar]
  32. Metuzals J., Mushynski W. E. Electron microscope and experimental investigations of the neurofilamentous network in Deiters' neurons. Relationship with the cell surface and nuclear pores. J Cell Biol. 1974 Jun;61(3):701–722. doi: 10.1083/jcb.61.3.701. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Nicklas W. J., Puszkin S., Berl S. Effect of vinblastine and colchicine on uptake and release of putative transmitters by synaptosomes and on brain actomyosin-like protein. J Neurochem. 1973 Jan;20(1):109–121. doi: 10.1111/j.1471-4159.1973.tb12109.x. [DOI] [PubMed] [Google Scholar]
  34. PALAY S. L. The morphology of synapses in the central nervous system. Exp Cell Res. 1958;14(Suppl 5):275–293. [PubMed] [Google Scholar]
  35. Pfenninger K. H. The cytochemistry of synaptic densities. I. An analysis of the bismuth lodide impregnation method. J Ultrastruct Res. 1971 Jan;34(1):103–122. doi: 10.1016/s0022-5320(71)90007-4. [DOI] [PubMed] [Google Scholar]
  36. Pfenninger K. H. The cytochemistry of synaptic densities. II. Proteinaceous components and mechanism of synaptic connectivity. J Ultrastruct Res. 1971 Jun;35(5):451–475. doi: 10.1016/s0022-5320(71)80005-9. [DOI] [PubMed] [Google Scholar]
  37. Rash J. E., Ellisman M. H. Studies of excitable membranes. I. Macromolecular specializations of the neuromuscular junction and the nonjunctional sarcolemma. J Cell Biol. 1974 Nov;63(2 Pt 1):567–586. doi: 10.1083/jcb.63.2.567. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. SAWANT P. L., SHIBKO, KUMTA U. S., TAPPEL A. L. ISOLATION OF RAT-LIVER LYSOSOMES AND THEIR GENERAL PROPERTIES. Biochim Biophys Acta. 1964 Apr 6;85:82–92. doi: 10.1016/0926-6569(64)90169-5. [DOI] [PubMed] [Google Scholar]
  39. Shelanski M. L., Gaskin F., Cantor C. R. Microtubule assembly in the absence of added nucleotides. Proc Natl Acad Sci U S A. 1973 Mar;70(3):765–768. doi: 10.1073/pnas.70.3.765. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Singer S. J. The molecular organization of membranes. Annu Rev Biochem. 1974;43(0):805–833. doi: 10.1146/annurev.bi.43.070174.004105. [DOI] [PubMed] [Google Scholar]
  41. TANAKA R., ABOOD L. G. PHOSPHOLIPID REQUIREMENT OF NA+,K+-ACTIVATED ADENOSINE TRIPHOSPHATASE FROM RAT BRAIN. Arch Biochem Biophys. 1964 Oct;108:47–52. doi: 10.1016/0003-9861(64)90353-4. [DOI] [PubMed] [Google Scholar]
  42. Thoa N. B., Wooten G. F., Axelrod J., Kopin I. J. Inhibition of release of dopamine- -hydroxylase and norepinephrine from sympathetic nerves by colchicine, vinblastine, or cytochalasin-B (hypogastric nerve stimulation-exocytosis-microtubules-microfilaments-guinea pig). Proc Natl Acad Sci U S A. 1972 Feb;69(2):520–522. doi: 10.1073/pnas.69.2.520. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. VERITY M. A., BROWN W. J. MURINE HEPATIC AND CEREBRAL LYSOSOMAL ACID PHOSPHOMONOESTERASES. Exp Cell Res. 1964 Jun;35:84–99. doi: 10.1016/0014-4827(64)90074-6. [DOI] [PubMed] [Google Scholar]
  44. Walters B. B., Matus A. I. Proteins of the synaptic junction. Biochem Soc Trans. 1975;3(1):109–112. doi: 10.1042/bst0030109. [DOI] [PubMed] [Google Scholar]
  45. Walters B. B., Matus A. I. Tubulin in postynaptic junctional lattice. Nature. 1975 Oct 9;257(5526):496–498. doi: 10.1038/257496a0. [DOI] [PubMed] [Google Scholar]

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

RESOURCES