Skip to main content

Advertisement

Springer Nature Link
Log in

De novo synthesis of NGF subunits in S-180 mouse sarcoma cell line

  • Original Articles
  • Published:
Neurochemical Research Aims and scope Submit manuscript

Abstract

It is an accepted hypothesis that the nerve growth factor protein (NGF) plays an important role in the development of vertebrate sympathetic and sensory ganglia and has effects on some central neurons. The best known NGF species is that isolated from the mouse submaxillary gland, MSG-NGF. MSG-NGF can be isolated as a subunit containing protein, 7S-NGF, made up of three dissimilar subunits called alpha-, beta-, and gamma-NGF. Beta-NGF is the biologically active subunit and its synthesis in vivo and in vitro has been demonstrated. Less is known about the synthesis of the alpha- and gamma-NGF or the assembly of the subunits into the 7S complex. In order to develop a clonal model system for the study of NGF synthesis, processing and secretion, affinity chromatography techniques were applied to cell extracts of S180 mouse sarcoma, a cell line known to synthesize NGF. After incubating S180 cells in35S-Methionine, cell extracts were exposed to antibody directed against alpha-NGF, gamma-NGF or beta-NGF covalently bound to Sepharose beads in order to elute and characterize the desired NGF subunits. Parallel experiments using immunoabsorbed [35S]Methionine-beta-NGF were carried out in the presence or absence of excess NGF, in order to demonstrate the specificity of this procedure. Affinity chromatography with a substrate analogue to arginine ester bound to Sepharose beads was also used to isolate de novo synthesized gamma-NGF. We were able to show that the S180 line synthesized alpha-, beta-, and gamma-NGF indistiguishable from alpha-, beta-, and gamma-NGF isolated from mouse submaxillary gland in terms of antigenic and physicochemical properties, and biological and enzymatic activities. These results are consistent with the hypothesis that NGF is synthesized, assembled and secreted by a single cell type.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Subscribe and save

Springer+ Basic
€34.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price includes VAT (Germany)

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Explore related subjects

Discover the latest articles and news from researchers in related subjects, suggested using machine learning.

References

  1. Barklis, E., and Perez-Polo, J. R. 1981. S-180 cells secrete nerve growth factor protein similar to 7S-nerve growth factor. J. Neurosci. 6:21–36.

    Google Scholar 

  2. Beck, C. E., and Perez-Polo, J. R. 1982. Human beta-nerve growth factor does not crossreact with antibodies to mouse beta-nerve growth factor in a two-site radioimmunoassay. J. Neurosci. Res. 8(2–3):137–152.

    Google Scholar 

  3. Berger, E. A., and Shooter, E. M. 1978. Biosynthesis of beta nerve growth factor in mouse submaxillary glands. J. Biol. Chem. 253(3):804–810.

    Google Scholar 

  4. Blum, M., Beck, C. E., and Perez-Polo, J. R. 1980. Absence of arginine-esteropeptidase activity in human nerve growth factor, Abstracts of Soc. for Neurosc., 6:376.

    Google Scholar 

  5. Bradshaw, R. A. 1978. Nerve growth factor. Ann. Rev. Biochem. 47:191–216.

    Google Scholar 

  6. Bradshaw, R. A. 1983. What cloned genes can tell us about nerve growth factor. Nature 303:751.

    Google Scholar 

  7. Cohen, S., Levi-Montalcini, R., and Hamburger, V. 1954. A nerve growth-stimulating factor isolated from sarcomas 37 and 180. Proc. Natl. Acad. Sci. USA 40:1014–1018.

    Google Scholar 

  8. Dicou, d'Eleni, Dider, Wion, et Philippe Brachet 1983. Biologie Moleculaire—Le facteur neurotrope nerve growth factor (NGF) synthetise par les cellules murine L-929 n'est pas associe aux sous-unites alpha et gamma. C.R. Acad. Sci. Paris 297:III-38 523–525.

    Google Scholar 

  9. Dunbar, J. C., and Bradshaw, R. A. 1985. Nerve growth factor biosynthesis: isolation and characterization of a guinea pig prostate kallikrein. Jour. of Cellular Biochem. 29:309–319.

    Google Scholar 

  10. Ebendal, T., Olson, L., Seiger, A., and Hedlund, K-O. 1980. Nerve growth factors in the rat iris. Nature 286:25–28.

    Google Scholar 

  11. Furukawa, Y., Furukawa, S., and Satoyoshi, E. 1984. Nerve growth factor secreted by mouse heart cells in culture. Jour. of Biol. Chem. 259:2:1259–1264.

    Google Scholar 

  12. Greene, L. A., Shooter, E. M., and Varon, S. 1969. Subunit interaction and enzymatic activity of mouse 7S nerve growth factor. Biochem. Jnr. 8:3735–3741.

    Google Scholar 

  13. Hendry, I. A., Stach, R., and Herrup, K. 1974. Characteristics of the retrograde axonal transport system for nerve growth factor in the sympathetic nervous system. Brain Res. 82:117–128.

    Google Scholar 

  14. Hoffman, H.-D., and Unsicker, K. 1982. The seminal vesicle of the bull: A new and very rich source of nerve growth factor. Eur. J. Biochem. 128:421–426.

    Google Scholar 

  15. Isackson, P. J., Bradshaw, R. A., and Jones, E. G. 1986. Expression of mouse nerve growth factor subunits. Soc. Neurosci. Abstract.

  16. Ishii, D. N., and Shooter, E. M. 1975. Regulation of nerve growth factor synthesis in mouse submaxillary glands by testosterone. J. Neurochem. 25:843–851.

    Google Scholar 

  17. Kim, I. S., and Pantazis, N. J. 1985. Biological and Biochemical properties of nerve growth factor synthesized by mouse S-180 cells in culture. Experimental Cell Res. 156:391–404.

    Google Scholar 

  18. Murphy, R. A., Oger, J., Saide, J. D., Blanchard, M. H., Arnason, G. W., Hogan, C., Pantazis, N. J., and Young, M. 1974. Secretion of nerve growth factor by central nervous system glioma cells in culture. J. Cell Biol. 72:769–773.

    Google Scholar 

  19. Mowry, M. D., Jensen, R. J., and Pantazis, N. J. 1984. Immunocytochemical localization and concentrations of the alpha and gamma subunits of 7S-nerve growth factor in the submandibular gland of the mouse. Cell and Tissue Res. 283:627–633.

    Google Scholar 

  20. Nagaiah, K., Lakshmann, J., Montgomery, P. P., Yu, M. W., and Guroff, G. 1978. Characteristics of the purified nerve growth factor antibody. J. Neurochem. 31:647–655.

    Google Scholar 

  21. Ouchterlony, O. 1953. Antigen-antibody reactions in gels. IV. Types of reactions in coordinated systems of diffusion. Acta Pathol Microbiol Scand 32:231–240.

    Google Scholar 

  22. Pantazis, N. J., Blanchard, M. H., Arnason, B. G. W., and Young, M. 1977. Molecular properties of the nerve growth factor secreted by L cells. Proc. Natl. Acad. Sci. USA 74(4):1492–1496.

    Google Scholar 

  23. Pantazis, N. J. 1983. Nerve growth factor synthesized by mouse fibroblast cells in culture: Absence of alpha and gamma subunits. Biochem. 22:4264–4271.

    Google Scholar 

  24. Perez-Polo, J. R., and Shooter, E. M. 1974. Subunit affinities in the 7S nerve growth factor complex. Neurobiol. 4:197–209.

    Google Scholar 

  25. Perez-Polo, J. R., and Shooter, E. M. 1975. The preparation and properties of nerve growth factor protein at allaline pH. Neurobiol. 5:329–338.

    Google Scholar 

  26. Perez-Polo, J. R., Hall, K., Livingston, K., and Westund, K. 1977. Steroid induction of nerve growth factor synthesis in cell culture. Life Sci. 21:1535–1544.

    Google Scholar 

  27. Perez-Polo, J. R., Bomar, H., Beck, C., and Hall, K. 1978. Nerve growth factor fromCrotalus adamenteus snake venom. J. Biol. Chem. 253(17):6140–6148.

    Google Scholar 

  28. Perez-Polo, J. R., Beck, C., Reynolds, C. P., and Blum, M. 1982. Human nerve growth factor: Comparative aspects, Chap. 2, pp. 129–163, in Growth and Maturation Factors (Guroff, G., ed.) John Wiley & Sons, Inc., New York.

    Google Scholar 

  29. Perez-Polo, J. R., Schulze, I., and Werrbach-Perez, K. 1983. The synthesis of nerve growth factor, in: Neurology and Neurobiology, Vol. 4, pp. 285–295, in Clinical and Biological Aspects of Peripheral Nerve Diseases (Battistin, L., Hashim, G. A., and Lajtha, A., eds.), Alan R. Liss, Inc, New York.

    Google Scholar 

  30. Ronne, H., Anundi, H., Rask, L., and Peterson, P. A. 1984. 7S nerve growth factor alpha and gamma subunits are closely related proteins. Biochem. 23:1229–1234.

    Google Scholar 

  31. Rubin, J. S., and Bradshaw, R. A. 1982. Isolation and partial amino acid sequence analysis of nerve growth factor from the guinea pig prostate. J. Neurosci. Res. 6:451–464.

    Google Scholar 

  32. Stach, R. W., and Shooter, E. M. 1980. Cross-linked 7S nerve growth factor is biologically inactive. J. Neurochem. 34(6):1499–1505.

    Google Scholar 

  33. Stoeckel, K., Gagnon, C., Guroff, G., and Thoenen, H. 1976. Purification of nerve growth factor antibodies by affinity chromatography. J. of Neurochem. 26:1207–1211.

    Google Scholar 

  34. Thomas, K. A., Baglan, N. C., and Bradshaw, R. A. 1981. The amino acid sequence of the gamma-subunit of mouse submaxillary gland 7S nerve growth factor. J. Biol. Chem. 256(17):9156–9166.

    Google Scholar 

  35. Varon, S., Nomura, J., and Shooter, E. M. 1967. The isolation of the mouse nerve growth factor protein in a high molecular weight form. Biochem. J. 6(7):2202–2209.

    Google Scholar 

  36. Varon, S., Nomura, J., Perez-Polo, J. R., and Shooter, E. M. 1972. The isolation and assay of the nerve growth factor proteins in Fried, R., (ed.) Methods in Neurochemistry 3(4), M. Dekker, New York.

    Google Scholar 

  37. Yankner, B. A., and Shooter, E. M. 1982. The biology and mechanism of action of nerve growth factor. Ann. Rev. Biochem. 51:845–868.

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Human Biological Chemistry and Genetics, The University of Texas Medical Branch, 77550, Galveston, Texas

    Karin Werrbach-Perez & J. Regino Perez-Polo

Authors
  1. Karin Werrbach-Perez
    View author publications

    Search author on:PubMed Google Scholar

  2. J. Regino Perez-Polo
    View author publications

    Search author on:PubMed Google Scholar

Additional information

Special Issue dedicated to Dr. E. M. Shooter and Dr. S. Varon.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Werrbach-Perez, K., Perez-Polo, J.R. De novo synthesis of NGF subunits in S-180 mouse sarcoma cell line. Neurochem Res 12, 875–883 (1987). https://doi.org/10.1007/BF00966309

Download citation

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00966309

Key Words