Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Liver phosphorylase kinase: characterization of two interconvertible forms and partial purification of phosphorylase kinase α

  • 18 Accesses

  • 14 Citations

Summary

The presence of two interconvertible forms of phosphorylase kinase has been confirmed in rat liver extracts. The pH optimum of the nonactivated form (PhK b) was lower than the pH optimum of the activated form (PhK α) as reported by others (2). In the absence of calcium the Km of PhK α for phosphorylase b was 53 + 10 U/ ml with a Vm of 17 = 1 U/gm of tissue. The Km of PhK α for phosphorylase b was 20 + 2 U/ml with a Vm of 65 U/gm. Calcium stimulated both forms of phosphorylase kinase(A0.5 ∼ 0.03 μM). In the presence of 0.1 μM calcium the Km for phosphorylase b of both forms of the enzyme was reduced. In addition, calcium increased the Vm of both forms, but the effect was greater for PhK b than for PhK α. The Km of both forms of phosphorylase kinase for ATP was 0.05 mM and was unaffected by calcium. All of these studies were done using liver phosphorylase b as substrate. Conditions for assaying PhK α activity virtually independent of PhK b activity also are indicated. This will enable the monitoring of interconversion reactions in tissue extracts.

Phosphorylase kinase a was purified to near homogeneity using DEAE-cellulose, Sepharose 4B gel filtration and ATP affinity chromatography. The molecular weight was approximately 1 × 106. The pII profile, calcium requirements and kinetic constants were the same as those for PhK a in the crude extract.

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

References

  1. 1.

    Wosilait, W. D. & Sutherland, E. W., 1956. J. Biol. Chem. 218: 469–481.

  2. 2.

    Vandenheede, J. R., Keppens, S. & DeWulf, H., 1977. Biochim. Biophys. Acta 481: 463–470.

  3. 3.

    Shimazu, T. & Amakawa, A., 1975. Biochim. Biophys. Acta 385: 242–256.

  4. 4.

    Khoo, J. C. & Steinberg, D., 1975. FEBS Letters 57: 68–72.

  5. 5.

    van de Werve, G., Hue, L. & Hers, H., 1977. Biochem. J. 162: 135–142.

  6. 6.

    Doorneweerd, D. D., Tan, A. W. H. & Nuttall, F. Q., 1979. Fed. Proc. 38: 718.

  7. 7.

    Appleman, M. M., Krebs, E. G. & Fischer, E. H., 1966. Biochem. 5: 2101–2107.

  8. 8.

    Tan, A. W. H. & Nuttall, F. Q., 1975. Biochim. Biophys. Acta 410: 45–60.

  9. 9.

    Killilea, S. D. & Whelan, W. J., 1976. Biochemistry 15, 1349–1356.

  10. 10.

    Walsh, D. A., Ashby, C. D., Gonzales, C., Calkins, D., Fischer, E. H. & Krebs, E. G., 1971. J. Biol. Chem. 246: 1977–1985.

  11. 11.

    Lowry, O. H., Roseborough, A. L., Farr, A. L. & Randall, R. J., 1951. J. Biol. Chem. 193: 265–275.

  12. 12.

    Zak, B. & Cohen, J., 1961. Clin. Chim. Acta 6: 665–670.

  13. 13.

    Vandenheede, J. R., Khandelwal, R. L. & Krebs, E. G., 1977. J. Biol. Chem. 252: 7488–7494.

  14. 14.

    Vandenheede, J. R., Keppens, S. & DeWulf, H., 1976. FEBS Letters 61: 213–217.

  15. 15.

    Portzehl, H., Caldwell, P. C. & Reugg, J. C., 1964. Biochim. Biophys. Acta 79: 581–591.

  16. 16.

    Krebs, E. G., Love, D. S., Bratvold, G. E., Trayer, K. S., Meyer, W. L. & Fischer, E. H., 1964. Biochemistry 3: 1022–1033.

  17. 17.

    Heilmeyer, L. M. G., Jr., Meyer, F., Haschke, R. H. & Fischer, E. H., 1970. J. Biol. Chem. 245: 6649–6656.

  18. 18.

    Brostrom, C. O., Hunkeler, F. L. & Krebs, E. G., 1971. J. Biol. Chem. 246: 1961–1967.

  19. 19.

    Keppens, S., Vandenheede, J. R. & DeWulf, H., 1977. Biochim. Biophys. Acta 496: 448–457.

  20. 20.

    Assimacopoulos-Jeannet, F. D., Blackmore, P. F. & Exton, J. H., 1977. J. Biol. Chem. 252: 2662–2669.

  21. 21.

    Hers, H., 1976. In Annual Review of Biochemistry (Snell, E. E., ed.), Vol. 45, pp. 167–189, Annual Reviews, Inc., Palo Alto, California.

  22. 22.

    Cohen, P., 1973. Eur. J. Biochem. 34: 1–14.

  23. 23.

    van de Werve, G. & Hers, H., 1979. Biochem. J. 178: 119–126.

  24. 24.

    Sakai, K., Matsumura, S., Okimura, Y., Hamamura, H. & Nishizuka, Y., 1979. J. Biol. Chem. 254: 6631–6637.

  25. 25.

    Vandenheede, J. R., DeWulf, H. & Merlevede, W., 1979. Eur. J. Biochem. 101: 51–58.

  26. 26.

    Chrisman, T. D. & Exton, J. H., 1980. J. Biol. Chem. 255, 3270–3273.

  27. 27.

    Wolf, D. P., Fischer, E. H. & Krebs, E. G., 1970. Biochem. 9: 1923–1929.

Download references

Author information

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Doorneweerd, D.D., Tan, A.W.H. & Nuttalll, F.Q. Liver phosphorylase kinase: characterization of two interconvertible forms and partial purification of phosphorylase kinase α. Mol Cell Biochem 47, 45–53 (1982). https://doi.org/10.1007/BF00241565

Download citation

Keywords

  • Phosphorylase
  • Kinetic Constant
  • Partial Purification
  • Liver Extract
  • Phosphorylase Kinase