Journal of Protein Chemistry

, Volume 12, Issue 6, pp 667–676 | Cite as

Immunological and biochemical study on tissue and subcellular distributions of protein kinase FA (an activating factor of ATP.Mg-dependent protein phosphatase): A simplified and efficient procedure for high quantity purification from brain

  • Jau-Song Yu
  • Shiaw-Der Yang
Article

Abstract

Although protein kinase FA/GSK-3α (an activating factor of ATP.Mg-dependent protein phosphatase) has been established as a cytosolic enzyme in mammalian nonnervous tissues involved in the metabolic regulation, immunological and biochemical studies on tissue and subcellular distributions demonstrate that kinase FA/GSK-3α is in fact a membrane-associated enzyme and most abundantly exists in brain particulate membrane fractions depending on the tissue homogenization conditions. For instance, when brain was homogenized in Polytron without 0.32M sucrose, approximately 40% of the total FA/GSK-3α was found in the cytosol. However, when brain was homogenized in buffer containing 0.32M sucrose and in a glass homogenizer with Teflon pestle, more than 80% of the total FA/GSK-3α was found associated with the particulate membrane fractions. By manipulating these findings, we have developed a simplified procedure for purification of homogeneous kinase FA/GSK-3α in high recovery and in a substantial amount from brain tissue. The data explain why kinase FA/GSK-3α cannot be isolated in a reasonable amount from most mammalian tissues for the past years. The specific pure antibody that can specifically recognize kinase FA/GSK-3α from crude tissue extracts together with the high quantity purification of the enzyme as presented in this report provides an initial key step for studies on the role of kinase FA/GSK-3α in the regulation of brain functions especially in the brain particulate membrane fractions.

Key words

Brain kinase FA affinity-purified antibody tissue and subcellular distribution purification 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Boyle, W. J., Smeal, T., Defize, L. H. K., Angel, P., Woodgett, J. R., Karin, M., and Hunter, T. (1991).Cell 64 573–584.Google Scholar
  2. Cohen, P. (1973).Eur. J. Biochem. 34 1–14.Google Scholar
  3. Dent, P., Campbell, D. G., Hubbard, M. J., and Cohen, P. (1989).FEBS Lett. 248 67–72.Google Scholar
  4. Depaoli-Roach, A. A. (1984).J. Biol. Chem. 259 12,144–12,152.Google Scholar
  5. Fiol, C. J., Haseman, J. H., Wang, Y., Roach, P. J., Roeske, R. W., Kowulczuk, M., and Depaoli-Roach, A. A. (1988).Arch. Biochem. Bipphys. 267 797–802.Google Scholar
  6. Fischer, E. H., and Krebs, E. G. (1958).J. Biol. Chem. 231 65–71.Google Scholar
  7. Goris, J., Defreyn, G., and Merlevede, W. (1979),FEBS Lett. 99 279–282.Google Scholar
  8. Guan, R., Khatra, B. S., and Cohlberg, J. A. (1991).J. Biol. Chem. 266 8262–8267.Google Scholar
  9. Hanker, D. P., Hughes, K., Woodgett, J. R., Brion, J.-P., and Anderton, B. H. (1992).Neurosci. Lett. 147 58–62.Google Scholar
  10. Hemmings, B. A., Yellowlees, D., Kernohan, J. C., and Cohen, P. (1981).Eur. J. Biochem. 119 443–451.Google Scholar
  11. Hemmings, B. A., Aitken, A., Cohen, P., Rymond, M., and Hofmann, F. (1982).Eur. J. Biochm. 127 473–481.Google Scholar
  12. Hughes, K., Ramacrishna, S., Benjamin, W. B., and Woodgett, J. R. (1992).Biochem. J.,288 309–314.Google Scholar
  13. Hughes, K., Nikolakaki, E., Plyte, S. E., Totty, N. F., and Woodgett, J. R. (1993).EMBO J. 12 803–808.Google Scholar
  14. Jurgensen, S., Shacter-Noiman, E., Huang, C. Y., Chock, P. B., Yang, S.-D., Vandenheede, J. R., and Merlevede, W. (1984).J. Biol. Chem. 259 5864–5870.Google Scholar
  15. Krebs, E. G., Kent, A. B., and Fischer, E. H. (1958).J. Biol. Chem. 231 73–83.Google Scholar
  16. Laemmli, U. K. (1970).Nature 227 680–685.Google Scholar
  17. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951).J. Biol. Chem. 193 265–275.Google Scholar
  18. Mackie, K., Sorkin, B. C., Nairn, A. C., Greengard, P., Edelman, G. M., and Cunningham, B. A. (1989).J. Neurosci. 9 1883–1896.Google Scholar
  19. Mandelkow, E.-M., Drewes, G., Biernat, J., Gustke, N., van Lint, J., Vandenheede, J. R., and Mandelkow, E. (1992).FEBS Lett. 314 315–321.Google Scholar
  20. Merlevede, W., Vandenheede, J. R., Goris, J., and Yang, S.-D. (1984).Curr. Top. Cell. Regul. 23 177–215.Google Scholar
  21. Reichlin, M. (1980).Methods Enzymol. 70 159–165.Google Scholar
  22. Taniuchi, M., Johnson, E. M., Roach, P. J., and Lawrence, J. C., Jr. (1986).J. Biol. Chem. 261 11,786–11,791.Google Scholar
  23. Tung, H. Y. L., and Reed, L. J. (1989).J. Biol. Chem. 264 2985–2990.Google Scholar
  24. Van Lint, J., Khandelwal, R. J., Merlevede, W., and Vandenheede, J. (1993).Anal. Biochem. 208 132–137.Google Scholar
  25. Vandenheede, J. R., Yang, S.-D., Goris, J., and Merlevede, W. (1980).J. Biol. Chem. 255 11,768–11,774.Google Scholar
  26. Woodgett, J. R. (1989).Anal. Biochem. 180 237–241.Google Scholar
  27. Woodgett, J. R. (1990).EMBO J. 9 2431–2438.Google Scholar
  28. Woodgett, J. R., and Cohen, P. (1984).Biochem. Biophys. Act. 788 339–347.Google Scholar
  29. Yang, S.-D., Vandenheede, J. R., Goris, J., and Merlevede, W. (1980).J. Biol. Chem. 255 11,759–11,767.Google Scholar
  30. Yang, S.-D., and Fong, Y.-L. (1985).J. Biol. Chem. 260 13,464–13,470.Google Scholar
  31. Yang, S.-D. (1986).J. Biol. Chem. 261 11,786–11,791.Google Scholar
  32. Yang, S.-D., Ho, L.-T., and Fung, T.-J. (1988).Biochem. Biophys. Res. Commun. 151 61–69.Google Scholar
  33. Yang, S.-D., Chou, C.-K., Huang, M., Song, J.-S., and Chen, H.-C. (1989).J. Biol. Chem. 264 5407–5411.Google Scholar
  34. Yang, S.-D., Yu, J.-S., and Lai, Y.-G. (1991).J. Prot. Chem. 10 171–181.Google Scholar
  35. Yang, S.-D., Song, J.-S., Hsieh, Y.-T., Liu, H.-W., and Chan, W.-H. (1992).J. Prot. Chem. 11 539–546.Google Scholar

Copyright information

© Plenum Publishing Corporation 1993

Authors and Affiliations

  • Jau-Song Yu
    • 1
    • 2
  • Shiaw-Der Yang
    • 1
    • 2
  1. 1.Institute of Biomedical ScienceNational Tsing Hua UniversityHsinchuTaiwan, Republic of China
  2. 2.Institute of Basic MedicineChang Gung Medical CollegeTao-YuanTaiwan, Republic of China

Personalised recommendations