Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1985 Nov 1;101(5):1977–1989. doi: 10.1083/jcb.101.5.1977

Nerve growth cones isolated from fetal rat brain. IV. Preparation of a membrane subfraction and identification of a membrane glycoprotein expressed on sprouting neurons

PMCID: PMC2113944  PMID: 3902858

Abstract

This study describes the preparation of a membrane subfraction from isolated nerve growth cone particles (GCPs) (see Pfenninger, K. H., L. Ellis, M. P. Johnson, L. B. Friedman, and S. Somlo, 1983, Cell, 35:573- 584) and the identification in this fraction of a glycoprotein expressed during neurite growth. While approximately 40 major polypeptides are visible in Coomassie Blue-stained SDS polyacrylamide gels of pelleted (partially disrupted) GCPs, a salt-washed membrane fraction prepared from lysed, detergent-permeabilized GCPs contains only 14% of this protein and has an unusually simple polypeptide pattern of seven major bands. Monoclonal antibodies have been generated to GCP membranes isolated from fetal rat brain. These antibodies have been screened differentially with synaptosomes from adult rat brain in order to identify those which recognize antigens expressed selectively during neurite growth. One such antibody (termed 5B4) recognizes a developmentally regulated membrane glycoprotein that is enriched in GCP membranes and expressed in fetal neurons sprouting in vitro. The 5B4 antigen in fetal brain migrates in SDS polyacrylamide gels as a diffuse band of approximately 185-255 kD, is rich in sialic acid, and consists of a small family of isoelectric variants. Freezing-thawing and neuraminidase digestion result in the cleavage of the native antigen into two new species migrating diffusely around 200 and 160 kD. Prolonged neuraminidase digestion sharpens these bands at about 180 and 135 kD, respectively. In the mature brain, antibody 5B4 recognizes a sparse polypeptide migrating at approximately 140 kD. As shown in the following paper (Wallis, I., L. Ellis, K. Suh, and K. H. Pfenninger, 1985, J. Cell Biol., 101:1990-1998), the fetal antigen is specifically associated with regions of neuronal sprouting and, therefore, can be used as a molecular marker of neurite growth.

Full Text

The Full Text of this article is available as a PDF (2.8 MB).

Selected References

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

  1. AMES B. N., DUBIN D. T. The role of polyamines in the neutralization of bacteriophage deoxyribonucleic acid. J Biol Chem. 1960 Mar;235:769–775. [PubMed] [Google Scholar]
  2. Bottenstein J. E., Sato G. H. Growth of a rat neuroblastoma cell line in serum-free supplemented medium. Proc Natl Acad Sci U S A. 1979 Jan;76(1):514–517. doi: 10.1073/pnas.76.1.514. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  4. Bunge M. B. Fine structure of nerve fibers and growth cones of isolated sympathetic neurons in culture. J Cell Biol. 1973 Mar;56(3):713–735. doi: 10.1083/jcb.56.3.713. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Campenot R. B. Local control of neurite development by nerve growth factor. Proc Natl Acad Sci U S A. 1977 Oct;74(10):4516–4519. doi: 10.1073/pnas.74.10.4516. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Chuong C. M., McClain D. A., Streit P., Edelman G. M. Neural cell adhesion molecules in rodent brains isolated by monoclonal antibodies with cross-species reactivity. Proc Natl Acad Sci U S A. 1982 Jul;79(13):4234–4238. doi: 10.1073/pnas.79.13.4234. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cohen R. S., Blomberg F., Berzins K., Siekevitz P. The structure of postsynaptic densities isolated from dog cerebral cortex. I. Overall morphology and protein composition. J Cell Biol. 1977 Jul;74(1):181–203. doi: 10.1083/jcb.74.1.181. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. De Cerro M. P., Snider R. S. Studies on the developing cerebellum. Ultrastructure of the growth cones. J Comp Neurol. 1968 Jul;133(3):341–362. doi: 10.1002/cne.901330305. [DOI] [PubMed] [Google Scholar]
  10. Ellis L., Katz F., Pfenninger K. H. Nerve growth cones isolated from fetal rat brain. II. Cyclic adenosine 3':5'-monophosphate (cAMP)-binding proteins and cAMP-dependent protein phosphorylation. J Neurosci. 1985 Jun;5(6):1393–1401. doi: 10.1523/JNEUROSCI.05-06-01393.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Engvall E., Perlmann P. Enzyme-linked immunosorbent assay, Elisa. 3. Quantitation of specific antibodies by enzyme-labeled anti-immunoglobulin in antigen-coated tubes. J Immunol. 1972 Jul;109(1):129–135. [PubMed] [Google Scholar]
  12. Finne J., Finne U., Deagostini-Bazin H., Goridis C. Occurrence of alpha 2-8 linked polysialosyl units in a neural cell adhesion molecule. Biochem Biophys Res Commun. 1983 Apr 29;112(2):482–487. doi: 10.1016/0006-291x(83)91490-0. [DOI] [PubMed] [Google Scholar]
  13. Finne J. Occurrence of unique polysialosyl carbohydrate units in glycoproteins of developing brain. J Biol Chem. 1982 Oct 25;257(20):11966–11970. [PubMed] [Google Scholar]
  14. Fujiki Y., Hubbard A. L., Fowler S., Lazarow P. B. Isolation of intracellular membranes by means of sodium carbonate treatment: application to endoplasmic reticulum. J Cell Biol. 1982 Apr;93(1):97–102. doi: 10.1083/jcb.93.1.97. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Giometti C. S., Anderson N. G., Tollaksen S. L., Edwards J. J., Anderson N. L. Analytical techniques for cell fractions. XXVII. Use of heart proteins as reference standards in two-dimensional electrophoresis. Anal Biochem. 1980 Feb;102(1):47–58. doi: 10.1016/0003-2697(80)90315-2. [DOI] [PubMed] [Google Scholar]
  17. Hasty D. L., Hay E. D. Freeze-fracture studies of the developing cell surface. II. Particle-free membrane blisters on glutaraldehyde-fixed corneal fibroblasts are artefacts. J Cell Biol. 1978 Sep;78(3):756–768. doi: 10.1083/jcb.78.3.756. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hawkes R., Niday E., Gordon J. A dot-immunobinding assay for monoclonal and other antibodies. Anal Biochem. 1982 Jan 1;119(1):142–147. doi: 10.1016/0003-2697(82)90677-7. [DOI] [PubMed] [Google Scholar]
  19. Hirn M., Ghandour M. S., Deagostini-Bazin H., Goridis C. Molecular heterogeneity and structural evolution during cerebellar ontogeny detected by monoclonal antibody of the mouse cell surface antigen BSP-2. Brain Res. 1983 Apr 11;265(1):87–100. doi: 10.1016/0006-8993(83)91337-9. [DOI] [PubMed] [Google Scholar]
  20. Hoffman S., Sorkin B. C., White P. C., Brackenbury R., Mailhammer R., Rutishauser U., Cunningham B. A., Edelman G. M. Chemical characterization of a neural cell adhesion molecule purified from embryonic brain membranes. J Biol Chem. 1982 Jul 10;257(13):7720–7729. [PubMed] [Google Scholar]
  21. Jockusch H., Jockusch B. M. Structural proteins in the growth cone of cultured spinal cord neurons. Exp Cell Res. 1981 Feb;131(2):345–352. doi: 10.1016/0014-4827(81)90237-8. [DOI] [PubMed] [Google Scholar]
  22. Jørgensen O. S., Delouvée A., Thiery J. P., Edelman G. M. The nervous system specific protein D2 is involved in adhesion among neurites from cultured rat ganglia. FEBS Lett. 1980 Feb 25;111(1):39–42. doi: 10.1016/0014-5793(80)80756-3. [DOI] [PubMed] [Google Scholar]
  23. Jørgensen O. S. Neuronal membrane D2-protein during rat brain ontogeny. J Neurochem. 1981 Oct;37(4):939–946. doi: 10.1111/j.1471-4159.1981.tb04481.x. [DOI] [PubMed] [Google Scholar]
  24. Jørgensen O. Polypeptides of the synaptic membrane antigens D1, D2, and D3. Biochim Biophys Acta. 1979 Nov 23;581(1):153–162. doi: 10.1016/0005-2795(79)90232-0. [DOI] [PubMed] [Google Scholar]
  25. Katz F., Ellis L., Pfenninger K. H. Nerve growth cones isolated from fetal rat brain. III. Calcium-dependent protein phosphorylation. J Neurosci. 1985 Jun;5(6):1402–1411. doi: 10.1523/JNEUROSCI.05-06-01402.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kim S. U., Hogue-Angeletti R., Gonatas N. K. Localization of nerve growth factor receptors in sympathetic neurons cultured in vitro. Brain Res. 1979 Jun 8;168(3):602–608. doi: 10.1016/0006-8993(79)90313-5. [DOI] [PubMed] [Google Scholar]
  27. Köhler G., Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature. 1975 Aug 7;256(5517):495–497. doi: 10.1038/256495a0. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Letourneau P. C. Immunocytochemical evidence for colocalization in neurite growth cones of actin and myosin and their relationship to cell--substratum adhesions. Dev Biol. 1981 Jul 15;85(1):113–122. doi: 10.1016/0012-1606(81)90240-2. [DOI] [PubMed] [Google Scholar]
  30. Marchisio P. C., Osborn M., Weber K. Changes in intracellular organization of tubulin and actin in N-18 neuroblastoma cells during the process of axon extension induced by serum deprivation. Brain Res. 1978 Oct 27;155(2):229–237. doi: 10.1016/0006-8993(78)91019-3. [DOI] [PubMed] [Google Scholar]
  31. Marchisio P. C., Osborn M., Weber K. The intracellular organization of actin and tubulin in cultured C-1300 mouse neuroblastoma cells (clone NB41A3). J Neurocytol. 1978 Oct;7(5):571–582. doi: 10.1007/BF01260890. [DOI] [PubMed] [Google Scholar]
  32. Margolis R. K., Margolis R. U. Distribution and characteristics of polysialosyl oligosaccharides in nervous tissue glycoproteins. Biochem Biophys Res Commun. 1983 Nov 15;116(3):889–894. doi: 10.1016/s0006-291x(83)80225-3. [DOI] [PubMed] [Google Scholar]
  33. Nestler E. J., Greengard P. Protein phosphorylation in the brain. Nature. 1983 Oct 13;305(5935):583–588. doi: 10.1038/305583a0. [DOI] [PubMed] [Google Scholar]
  34. O'Farrell P. H. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975 May 25;250(10):4007–4021. [PMC free article] [PubMed] [Google Scholar]
  35. Pfenninger K. H., Bunge R. P. Freeze-fracturing of nerve growth cones and young fibers. A study of developing plasma membrane. J Cell Biol. 1974 Oct;63(1):180–196. doi: 10.1083/jcb.63.1.180. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Pfenninger K. H., Ellis L., Johnson M. P., Friedman L. B., Somlo S. Nerve growth cones isolated from fetal rat brain: subcellular fractionation and characterization. Cell. 1983 Dec;35(2 Pt 1):573–584. doi: 10.1016/0092-8674(83)90191-5. [DOI] [PubMed] [Google Scholar]
  37. Pfenninger K. H., Johnson M. P. Membrane biogenesis in the sprouting neuron. I. Selective transfer of newly synthesized phospholipid into the growing neurite. J Cell Biol. 1983 Oct;97(4):1038–1042. doi: 10.1083/jcb.97.4.1038. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Pfenninger K. H., Maylié-Pfenninger M. F., Friedman L. B., Simkowitz P. Lectin labeling of sprouting neurons. III. Type-specific glycoconjugates on growth cones of different origin. Dev Biol. 1984 Nov;106(1):97–108. doi: 10.1016/0012-1606(84)90066-6. [DOI] [PubMed] [Google Scholar]
  39. Pfenninger K. H., Maylié-Pfenninger M. F. Lectin labeling of sprouting neurons. I. Regional distribution of surface glycoconjugates. J Cell Biol. 1981 Jun;89(3):536–546. doi: 10.1083/jcb.89.3.536. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Pfenninger K. H., Maylié-Pfenninger M. F. Lectin labeling of sprouting neurons. II. Relative movement and appearance of glycoconjugates during plasmalemmal expansion. J Cell Biol. 1981 Jun;89(3):547–559. doi: 10.1083/jcb.89.3.547. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Rees R. P., Reese T. S. New structural features of freeze-substituted neuritic growth cones. Neuroscience. 1981;6(2):247–254. doi: 10.1016/0306-4522(81)90060-9. [DOI] [PubMed] [Google Scholar]
  42. Rohrer H., Barde Y. A. Presence and disappearance of nerve growth factor receptors on sensory neurons in culture. Dev Biol. 1982 Feb;89(2):309–315. doi: 10.1016/0012-1606(82)90320-7. [DOI] [PubMed] [Google Scholar]
  43. Rothbard J. B., Brackenbury R., Cunningham B. A., Edelman G. M. Differences in the carbohydrate structures of neural cell-adhesion molecules from adult and embryonic chicken brains. J Biol Chem. 1982 Sep 25;257(18):11064–11069. [PubMed] [Google Scholar]
  44. Rougon G., Deagostini-Bazin H., Hirn M., Goridis C. Tissue- and developmental stage-specific forms of a neural cell surface antigen linked to differences in glycosylation of a common polypeptide. EMBO J. 1982;1(10):1239–1244. doi: 10.1002/j.1460-2075.1982.tb00019.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Schwartz M., Ernst S. A., Siegel G. J., Agranoff B. W. Immunocytochemical localization of (Na+, K+)-ATPase in the goldfish optic nerve. J Neurochem. 1981 Jan;36(1):107–115. doi: 10.1111/j.1471-4159.1981.tb02384.x. [DOI] [PubMed] [Google Scholar]
  46. Sharon J., Morrison S. L., Kabat E. A. Detection of specific hybridoma clones by replica immunoadsorption of their secreted antibodies. Proc Natl Acad Sci U S A. 1979 Mar;76(3):1420–1424. doi: 10.1073/pnas.76.3.1420. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Shaw G., Osborn M., Weber K. Arrangement of neurofilaments, microtubules and microfilament-associated proteins in cultured dorsal root ganglia cells. Eur J Cell Biol. 1981 Apr;24(1):20–27. [PubMed] [Google Scholar]
  48. Shaw G., Weber K. The distribution of the neurofilament triplet proteins within individual neurones. Exp Cell Res. 1981 Nov;136(1):119–125. doi: 10.1016/0014-4827(81)90043-4. [DOI] [PubMed] [Google Scholar]
  49. 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]
  50. Small R. K., Pfenninger K. H. Components of the plasma membrane of growing axons. I. Size and distribution of intramembrane particles. J Cell Biol. 1984 Apr;98(4):1422–1433. doi: 10.1083/jcb.98.4.1422. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. TOMARELLI R. M., CHARNEY J., HARDING M. L. The use of azoalbumin as a substrate in the colorimetric determination or peptic and tryptic activity. J Lab Clin Med. 1949 Mar;34(3):428–433. [PubMed] [Google Scholar]
  52. Tennyson V. M. The fine structure of the axon and growth cone of the dorsal root neuroblast of the rabbit embryo. J Cell Biol. 1970 Jan;44(1):62–79. doi: 10.1083/jcb.44.1.62. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Vimr E. R., McCoy R. D., Vollger H. F., Wilkison N. C., Troy F. A. Use of prokaryotic-derived probes to identify poly(sialic acid) in neonatal neuronal membranes. Proc Natl Acad Sci U S A. 1984 Apr;81(7):1971–1975. doi: 10.1073/pnas.81.7.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Wallis I., Ellis L., Suh K., Pfenninger K. H. Immunolocalization of a neuronal growth-dependent membrane glycoprotein. J Cell Biol. 1985 Nov;101(5 Pt 1):1990–1998. doi: 10.1083/jcb.101.5.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Weber K., Osborn M. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem. 1969 Aug 25;244(16):4406–4412. [PubMed] [Google Scholar]
  57. Wray W., Boulikas T., Wray V. P., Hancock R. Silver staining of proteins in polyacrylamide gels. Anal Biochem. 1981 Nov 15;118(1):197–203. doi: 10.1016/0003-2697(81)90179-2. [DOI] [PubMed] [Google Scholar]
  58. Yamada K. M., Spooner B. S., Wessells N. K. Ultrastructure and function of growth cones and axons of cultured nerve cells. J Cell Biol. 1971 Jun;49(3):614–635. doi: 10.1083/jcb.49.3.614. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES