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Neurochemical Research

, Volume 9, Issue 10, pp 1493–1507 | Cite as

Metabolic studies in vitro of the CNS cytoskeletal proteins: Synthesis and degradation

  • Marion E. Smith
  • Virginia Perret
  • Lawrence F. Eng
Article

Abstract

General aspects of metabolic features of the most prominent CNS intermediate filament proteins, the 200,000 (200K), 150,000 (150K), and 70,000 (70K) dalton proteins of the neuron, and the glial fibrillary acidic protein (GFAP) have been explored using the incubated spinal cord slice from the rat. Measurement of shortterm uptake of3H-labeled amino acids into the individual proteins separated on polyacrylamide gels revealed that of the three neurofilament proteins, 200K was most metabolically active, 150K was less active, and 70K contained very little incorporated radioactivity. Glial fibrillary acidic protein based on Coomassie blue stain affinity showed less metabolic activity than any of the neurofilament proteins. Those relationships were constant at all ages, but the metabolic activity of all CNS intermediate filaments decreased with age. When Ca2+ was present in the medium of the incubated slices, the intermediate filaments were rapidly destroyed, but GFAP was more resistant to degradation than the neurofilament proteins. GFAP and probably the neurofilament proteins also were relatively resistant to Ca2+-activated degradative mechanisms in spinal cords of rats at younger ages (15 day) than in those of older animals (10–18 months). It is likely that the Ca2+ activated protease is less active in developing animals in which the nerve tracts are still elongating, than in adults. These results suggest that GFAP is less active metabolically and more resistant to degradation than the neurofilament proteins at all stages of maturation, but that metabolic activity of all CNS intermediate filaments decreases with age while the susceptibility to degradation increases.

Keywords

Spinal Cord Metabolic Activity Glial Fibrillary Acidic Protein Coomassie Intermediate Filament 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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References

  1. 1.
    Norton, W. T., andGoldman, J. E. 1980. Neurofilaments. Pages 301–329,in Bradshaw, R. A., andSchneider, D. M. (eds.), Proteins of the Nervous System, Raven Press, New York.Google Scholar
  2. 2.
    Chiu, F.-C., Goldman, J. E., andNorton, W. T. 1983. Biochemistry of neurofilaments. Pages 27–56,in Marotta, C. A. (ed.) Neurofilaments, University of Minnesota Press, Minneapolis.Google Scholar
  3. 3.
    Eng, L. F. 1980. The glial fibrillary acidic protein. Pages 85–117,in Bradshaw, R. A., andSchneider, D. M. (eds.), Proteins of the Nervous System, Raven Press, New York.Google Scholar
  4. 4.
    Chiu, F.-C., Norton, W. T., andFields, K. L. 1981. The cytoskeleton of primary astrocytes in culture contains actin, glial fibrillary acidic protein and the fibroblast-type filament protein, vimentin. J. Neurochem. 37:147–155.PubMedGoogle Scholar
  5. 5.
    Lowry, O. H., Rosebrough, N. J., Farr, A. L., andRandall, R. J. 1951. Protein measurements with Folin phenol reagent. J. Biol. Chem. 193:265–275.PubMedGoogle Scholar
  6. 6.
    Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (Lond.) 227:680–685.Google Scholar
  7. 7.
    Smith, M. E., Somera, F. P., andEng, L. F. 1983. Immunocytochemical staining for glial fibrillary acidic protein and the metabolism of cytoskeletal proteins in experimental allergic encephalomyelitis. Brain Res. 264:241–253.PubMedGoogle Scholar
  8. 8.
    Chiu, F.-C., andNorton, W. T. 1982. Bulk preparation of CNS cytoskeletal and the separation of individual neurofilament proteins by gel filtration. Dye-binding characteristics and amino acid compositions. J. Neurochem. 39:1252–1260.PubMedGoogle Scholar
  9. 9.
    Smith, M. E. 1980. Biosynthesis of peripheral nervous system myelin proteins in vitro. J. Neurochem. 35:1183–1189.PubMedGoogle Scholar
  10. 10.
    Towbin, H., Staehelin, T., andGordon, J. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets. Procedure and some applications. Proc. Natl. Acad. Sci. USA 76:4350–4354.PubMedGoogle Scholar
  11. 11.
    Eng, L. F., andRubinstein, L. J. 1978. Contribution of immunohisto-chemistry to diagnostic problems of human cerebral tumors. J. Histochem. Cytochem. 25:513–522.Google Scholar
  12. 12.
    Sternberger, L. A. 1979. Immunocytochemistry. John Wiley and Sons, New York.Google Scholar
  13. 13.
    Aamondde, S. J., Fajardo, M., Naughton, S. A., andEng, L. F. 1983. Degradation of glial fibrillary acidic protein by a calcium dependent proteinase: An electroblot study. Brain Res. 262:275–282.PubMedGoogle Scholar
  14. 14.
    Ellisman, M. H., andPorter, K. R. 1983. Introduction to the cytoskeleton. Pages 3–26,in Marotta, C. A. (ed.), Neurofilaments, University of Minnesota Press, Minneapolis.Google Scholar
  15. 15.
    Cszosnek, H. P., Soifer, D., andWisniewski, H. M. 1980. Studies on the biosynthesis of neurofilament proteins. J. Cell Biol. 85:726–734.PubMedGoogle Scholar
  16. 16.
    Hoffman, P. M., andLasek, R. J. 1975. The slow component of axonal transport: Identification of major structural polypeptides of the axon and their generality among mammalian neurons. J. Cell Biol. 66:351–366.PubMedGoogle Scholar
  17. 17.
    Lasek, R. J., andBlack, M. M. 1977. How do axons stop growing? Some clues from the metabolism of the proteins in the slow component of axonal transport. Pages 161–169,in Roberts, S., Lajtha, A., andGispen, W. H. (eds.), Mechanisms, Regulation, and Special Functions of Protein Synthesis of the Brain, Elsevier, North Holland, Amsterdam.Google Scholar
  18. 18.
    Willard, M. 1983. Neurofilaments and axonal transport. Pages 86–116,in Marotta, C. A. (ed.), Neurofilaments, University of Minnesota Press, Minneapolis.Google Scholar
  19. 19.
    Shecket, G., andLasek, R. J. 1980. Preparation of neurofilament protein from guinea pig peripheral nerve and spinal cord. J. Neurochem. 35:1335–1344.PubMedGoogle Scholar
  20. 20.
    Guroff, G. 1964. A neutral calcium-activated proteinase from the soluble fraction of rat brain. J. Biol. Chem. 239:149–155.PubMedGoogle Scholar
  21. 21.
    Nixon, R. A. 1983. Proteolysis of neurofilaments. Pages 117–154,in Marotta, C. A. (ed.), Neurofilaments, University of Minnesota Press, Minneapolis.Google Scholar
  22. 22.
    Schlaepfer, W. W., andZimmerman, U.-J. P. 1981. Calcium-mediated breakdown of glial filaments and neurofilaments in rat optic nerve and spinal cord. Neurochem. Res. 6:243–255.PubMedGoogle Scholar
  23. 23.
    Bigbee, J. W., Bigner, D. D., Pegram, C., andEng, L. F. 1983. Study of glial fibrillary acidic protein in a human glioma cell line grown in culture and as a solid tumor. J. Neurochem. 40:460–467.PubMedGoogle Scholar
  24. 24.
    Chiu, F.-C. andGoldman, J. E. 1984. Synthesis and turnover of cytoskeletal proteins in cultured astrocytes. J. Neurochem. 42:166–174.PubMedGoogle Scholar

Copyright information

© Plenum Publishing Corporation 1984

Authors and Affiliations

  • Marion E. Smith
    • 1
    • 2
  • Virginia Perret
    • 1
    • 2
  • Lawrence F. Eng
    • 1
    • 2
  1. 1.Departments of Neurology and PathologyVeterans Administration Medical CenterPalo Alto
  2. 2.Stanford University School of MedicineStanford

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