Skip to main content
SpringerLink
Log in

Distribution of endogenously phosphorylated proteins in subcellular fractions of rat cerebral cortex

  • Published:
Neurochemical Research Aims and scope Submit manuscript

Abstract

The cerebral cortex from adult rats was separated into several subcellular fractions by using established methods of differential and sucrose density gradient centrifugation. Aliquots from each fraction were incubated with γ-32P-ATP, in the presence and absence of adenosine 3′,5′-monophosphate (cyclic AMP), and its protein constituents were separated by means of SDS-slab gel electrophoresis. Fractions containing nuclei, synaptosomes, myelin, microsomes, and soluble proteins each showed a characteristic pattern of protein staining and of endogenously phosphorylated proteins detected by autoradiography of the gels. Cyclic AMP-stimulated phosphorylation of proteins with MW 78K and 84K can serve as markers for membranes of synaptic origin, while cyclic AMP-independent phosphorylation of low-molecular-weight proteins (15K–20K) is characteristic of myelin. The finding of different phosphoproteins in various subcellular fractions may be related to the diversity of cellular functions known to be regulated by phosphorylative activity.

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

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Heald, P. J. 1957. The incorporation of phosphate into cerebral phosphoproteins promoted by electrical impulses. Biochem. J. 66:659–663.

    Google Scholar 

  2. Trevor, A. J., andRodnight, R. 1965. The subcellular localization of cerebral phosphoproteins sensitive to electrical stimulation. Biochem. J. 95:889–896.

    Google Scholar 

  3. Miyamoto, E., Kuo, J. F., andGreengard, P. 1969. Adenosine 3′,5′-monophosphate-dependent protein kinase from rat brain. Science 165:63–65.

    Google Scholar 

  4. Rall, T. W., andGilman, A. G. 1970. The role of cyclic AMP in the nervous system. Neurosci. Res. Progr. Bull. 8:221–323.

    Google Scholar 

  5. Greengard, P., andKebabian, J. W. 1974. Role of cyclic AMP in synaptic transmission in the mammalian peripheral nervous system. Fed. Proc. 33:1059–1067.

    Google Scholar 

  6. Williams, M., andRodnight, R. 1974. Evidence for a role for protein phosphorylation in synaptic function in the cerebral cortex mediated through a beta-noradrenergic receptor. Brain Res. 77:502–506.

    Google Scholar 

  7. Weller, M., andMorgan, I. G. A possible role of the phosphorylation of synaptic membrane proteins in the control of Ca++ permeability. Biochim. Biophys. Acta 465:527–534.

  8. Rodnight, R. 1975. Cyclic AMP and protein phosphorylation in the central nervous system in relation to synaptic function. Pages 205–228,in Berl, S., Clarke, D. D., andSchneider, D. (eds.), Metabolic Compartmentation and Neurotransmission: Relation to Brain Structure and Function, Plenum Press, New York.

    Google Scholar 

  9. Greengard, P. 1976. A possible role for cyclic nucleotides and phosphorylated membrane proteins in postsynaptic actions of neurotransmitters. Nature 260:101–108.

    Google Scholar 

  10. Rubin, C. S., andRosen, O. M. 1975. Protein phosphorylation. Ann. Rev. Biochem. 44:831–888.

    Google Scholar 

  11. Costa, E., Kurosawa, A., andGuidotti, A. 1976. Activation and nuclear translocation of protein kinase during transsynaptic induction of tyrosine 3-monooxygenase. Proc. Natl. Acad. Sci. USA 73:1058–1062.

    Google Scholar 

  12. Ehrlich, Y. H., Brunngraber, E. G., Sinha, P. K., andPrasad, K. N. 1977. Specific alterations in phosphorylation of cytosol proteins from differentiating neuroblastoma cells. Nature 265:238–240.

    Google Scholar 

  13. Ehrlich, Y. H., andRouttenberg, A. 1974. Cyclic AMP regulates phosphorylation of three protein components of rat cerebral cortex membranes for thirty minutes. FEBS Lett. 45:237–242.

    Google Scholar 

  14. Routtenberg, A., andEhrlich, Y. H. 1975. Endogenous phosphorylation of four cerebral cortical membrane proteins: Role of cyclic nucleotides, ATP, and divalent cations. Brain Res. 92:415–430.

    Google Scholar 

  15. Dutton, G. R., andMahler, H. R. 1968.In vitro RNA synthesis by intact rat brain nuclei. J. Neurochem. 15:765–780.

    Google Scholar 

  16. Whittaker, V. P. 1966. Some properties of synaptic membranes isolated from the central nervous system. Ann. NY Acad. Sci. 137:982–998.

    Google Scholar 

  17. Lowry, O. H., Rosebrough, N. J., Farr, A. L., andRandall, R. J. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 93:265–275.

    Google Scholar 

  18. Brunngraber, E. G., Aguilar, V., andOccomy, G. 1963. The intracellular distribution of glycolytic and tricarboxylic acid cycle enzymes in rat brain mitochondrial preparations. J. Neurochem. 10:433–438.

    Google Scholar 

  19. Koenig, H. 1969. Lysosomes. Pages 255–301,in Lajtha, A. (ed.), Handbook of Neurochemistry, Vol. II, Plenum Press, New York.

    Google Scholar 

  20. Grossfeld, R. M., andShooter, E. M. 1971. A study in the changes in protein composition of mouse brain during ontogenetic development. J. Neurochem. 18:2265–2277.

    Google Scholar 

  21. Cammer, W., andNorton, W. T. 1976. Disc gel electrophoresis of myelin proteins: New observations on development of the intermediate proteins (DM-20). Brain Res. 109:643–648.

    Google Scholar 

  22. Prasad, K. N. 1975. Differentiation of neuroblastoma cells in culture. Biol. Rev. 50:129–165.

    Google Scholar 

  23. Maeno, H., Johnson, M., andGreengard, P. 1971. Subcellular distribution of adenosine 3′,5′-monophosphate-dependent protein kinase in rat brain. J. Biol. Chem. 246:134–142.

    Google Scholar 

  24. Weller, M., andMorgan, I. 1976. Distribution of protein kinase activities in subcellular fractions of rat brain. Biochim. Biophys. Acta 436:675–685.

    Google Scholar 

  25. Uno, I., Ueda, T., andGreengard, P. 1976. Differences in properties of cytosol and membrane-derived protein kinases. J. Biol. Chem. 251:2192–2195.

    Google Scholar 

  26. Ueda, T., Rudolph, S. A., andGreengard, P. 1975. Solubilization of a phosphoprotein and its associated cyclic AMP-dependent protein kinase and phosphoprotein phosphatase from synaptic membrane fractions, and some kinetic evidence for their existance as a complex. Arch. Biochem. Biophys. 170:492–503.

    Google Scholar 

  27. Mahadik, S. P., Korenovsky, A., andRapport, M. M. 1976. Slab gel analysis of the polypeptide components of rat brain subcellular organelles. Anal. Biochem. 76:615–633.

    Google Scholar 

  28. Ueda, T., Maeno, H., andGreengard, P. 1973. Regulation of endogenous phosphorylation of specific proteins in synaptic membrane fractions from rat brain by adenosine 3′,5′-monophosphate. J. Biol. Chem. 248:8295–8305.

    Google Scholar 

  29. Malkinson, A. M., Krueger, B. K., Rudolph, S. A., Casnellie, J. E., Haley, B. E., andGreengard, P. 1975. Widespread occurrence of a specific protein in vertebrate tissues and regulation by cyclic AMP of its endogenous phosphorylation and dephosphorylation. Metabolism 24:331–341.

    Google Scholar 

  30. Ehrlich, Y. H., Rabjohns, R. R., andRouttenberg, A. 1977. Experiential input alters the phosphorylation of specific proteins in brain membranes. Biochem. Pharm. Behav. 6:169–174.

    Google Scholar 

  31. Ehrlich, Y. H., Brunngraber, E. G., andBonnet, K. A. 1977. Protein phosphorylation in neostriatal membranes after narcotic exposure. Trans. Am. Soc. Neurochem. 8:82.

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Biochemistry Laboratory Missouri Institute of Psychiatry School of Medicine, University of Missouri, Columbia, 5400 Arsenal Street, 63139, St. Louis, Missouri

    Yigal H. Ehrlich, Leonard G. Davis, Thomas Gilfoil & Eric G. Brunngraber

Authors
  1. Yigal H. Ehrlich
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Leonard G. Davis
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Thomas Gilfoil
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Eric G. Brunngraber
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Ehrlich, Y.H., Davis, L.G., Gilfoil, T. et al. Distribution of endogenously phosphorylated proteins in subcellular fractions of rat cerebral cortex. Neurochem Res 2, 533–548 (1977). https://doi.org/10.1007/BF00966013

Download citation

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00966013

Keywords

Search

Navigation