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Neurochemical Research

, Volume 7, Issue 2, pp 127–137 | Cite as

Specific proteolysis of a brain membrane phosphoprotein (B-50): Effects of calcium and calmodulin

  • H. Zwiers
  • W. H. Gispen
  • L. Kleine
  • H. R. Mahler
Original Articles

Abstract

The membrane bound phosphoprotein B-50 (MW 48K) was isolated from rat brain tissue. The fraction containing the highest endogenous B-50 phosphorylating activity (ASP 57–82%) contains protease activity. In the absence of calcium a time-dependent decrease of the protein B-50 is observed. Under these conditions another phosphoprotein B-60 (MW 46K) appears in the incubation medium. Addition of calcium and/or calmodulin enhances the protease activity whereas the substrate specificity is lost. Results of both isoelectric focussing and peptide mapping indicate that B-50 and B-60 are related proteins. These data support our hypothesis that the recently isolated behaviorally active peptide PIP (MW approx. 1600 D) is the smaller cleavage product of the proteolytic degradation of B-50 to B-60.

Keywords

Calcium Peptide Brain Tissue Substrate Specificity Protease Activity 
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.
    Zwiers, H., Tonnaer, J., Wiegant, V. M., Schotman, P., andGispen, W. H. 1979. ACTH-sensitive protein kinase from rat brain membranes. J. Neurochem. 33:247–256.PubMedGoogle Scholar
  2. 2.
    Zwiers, H., Schotman, P., andGispen, W. H. 1980. Purification and some characteristics of an ACTH-sensitive protein kinase and its substrate protein in rat brain membranes. J. Neurochem. 34:1689–1700.PubMedGoogle Scholar
  3. 3.
    Zwiers, H., Veldhuis, D., Schotman, P., andGispen, W. H. 1976. ACTH, cyclic nucleotides and brain protein phosphorylation in vitro. Neurochem. Res. 1:669–677.Google Scholar
  4. 4.
    Zwiers, H., Wiegant, V. M., Schotman, P., andGispen, W. H. 1978. ACTH-induced inhibition of endogenous rat brain protein phosphorylation in vitro: structure-activity. Neurochem. Res. 3:455–463.Google Scholar
  5. 5.
    Zwiers, H., Verhoef, J., Schotman, P., andGispen, W. H. 1980. A new phosphorylation-inhibiting peptide (PIP) with behavioral activity from rat brain membranes. FEBS Lett. 112:168–172.PubMedGoogle Scholar
  6. 6.
    Zwiers, H., Aloyo, V., andGispen, W. H. 1981. Behavioral and neurochemical effects of the new peptide dynorphin (1–13): comparison with other neuropeptides. Life Sci. 28:2545–2553.PubMedGoogle Scholar
  7. 7.
    Gispen, W. H., Zwiers, H., Wiegant, V. M., Schotman, P., andWilson, J. E. 1979. The behavioral active neuropeptide ACTH as neurohormone and neuromodulator: The role of cyclic nucleotides and membrane phosphoproteins. Adv. Exp. Med. and Biol. 116:199–224.Google Scholar
  8. 8.
    Mahler, H. R., Kleine, L. P., andSorensen, R. G. 1981. Topography of synaptic phosphoprotein. Trans. Am. Soc. Neurochem. 12:213.Google Scholar
  9. 9.
    Cleveland, D. W., Fischer, S. G., Kirschner, M. W., andLaemmli, U. K. 1977. Peptide mapping by limited proteolysis in sodium dodecyl sulphate and analysis by gel electrophoresis. J. Biol. Chem. 252:1102–1106.Google Scholar
  10. 10.
    Wallace, R. W., andCheung, W. Y. 1979. Calmodulin: Production of an antibody in rabbit and development of a radioimmunoassay. J. Biol. Chem. 254:6564–6571.PubMedGoogle Scholar
  11. 11.
    Lowry, O. H., Rosebrough, N. J., Farr, A. L., andRandall, R. J. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265–275.PubMedGoogle Scholar
  12. 12.
    Jolles, J., Zwiers, H., van Dongen, C., Schotman, P., Wirtz, K. W. A., andGispen, W. H. 1980. Modulation of brain polyphosphoinositide metabolism by ACTH-sensitive protein phosphorylation. Nature (Lond.) 286:623–625.Google Scholar
  13. 13.
    Weber, W., andHilz, H. 1979. Stoichiometry of cAMP binding and limited proteolysis of protein kinase regulatory subunits R I and R II. Biochem. Biophys. Res. Commun. 90:1073–1081.Google Scholar
  14. 14.
    Taki, Y., Kishimoto, A., Inoue, M., andNishizuka, Y. 1977. Studies on a cyclic nucleotide-independent protein kinase and its proenzyme in mammalian tissue I. J. Biol. Chem. 252:7603–7609.PubMedGoogle Scholar
  15. 15.
    Jolles, J., Aloyo, V., andGispen, W. H. 1981. Molecular correlates between pituitary hormones and behavior. Pages 285–316,in Brown, I. R. (ed.), Molecular approaches to neurobiology, Academic Press, New York.Google Scholar

Copyright information

© Plenum Publishing Corporation 1982

Authors and Affiliations

  • H. Zwiers
    • 1
  • W. H. Gispen
    • 1
  • L. Kleine
    • 2
  • H. R. Mahler
    • 2
  1. 1.Division of Molecular Neurobiology Rudolf Magnus Institute for Pharmacology Institute of Molecular BiologyUniversity of UtrechtUtrechtThe Netherlands
  2. 2.Department of ChemistryIndiana UniversityBloomingtonUSA

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