Skip to main content
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1981 Feb;78(2):871–874. doi: 10.1073/pnas.78.2.871

Mechanism of activation of cyclic nucleotide phosphodiesterase: requirement of the binding of four Ca2+ to calmodulin for activation.

C Y Huang, V Chau, P B Chock, J H Wang, R K Sharma
PMCID: PMC319905  PMID: 6262778

Abstract

Kinetic studies on the activation of cyclic nucleotide phosphodiesterase (3',5'-cyclic-nucleotide 5'-nucleotidohydrolase, EC 3.1.4.17) as a function of calmodulin and Ca2+ concentrations have been carried out. A general approach to analyzing the mechanism of activation, which takes into consideration the various interactions among phosphodiesterase and calmodulin liganded with Ca2+ to differing degrees, is presented. The method is applicable to other calmodulin-regulated enzyme systems. Our kinetic analysis reveals that all four Ca2+ must be bound to calmodulin for the protein to form an activated complex with phosphodiesterase. The mechanistic and regulatory advantages of having four Ca2+ sites on calmodulin can be briefly stated as follows. (i) With the enzyme--calmodulin--Ca4(2+) complex as the dominant active species, the activation of phosphodiesterase as a function of Ca2+ concentration is highly cooperative. This phenomenon serves as an effective on/off switch for phosphodiesterase activation. (ii) At normal cellular levels of Ca2+ (less than 0.1 microM), phosphodiesterase and calmodulin do not form a complex. Thus, the distribution of calmodulin among its various target enzymes is reshuffled for each Ca2+ surge. (iii) The affinity between the enzyme and the fully liganded calmodulin (0.1-1 mM) is 10(4)-10(5) times better than that in the absence of Ca2+ (greater than or equal to 10 microM). The tremendous increase in affinity can be achieved rather easily through a 10- to 20-fold increase in the affinity of Ca2+ for the enzyme-calmodulin complex in each of the four binding steps.

Full text

PDF
871

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Brostrom C. O., Wolff D. J. Calcium-dependent cyclic nucleotide phosphodiesterase from glial tumor cells. Arch Biochem Biophys. 1974 Dec;165(2):715–727. doi: 10.1016/0003-9861(74)90300-2. [DOI] [PubMed] [Google Scholar]
  2. Cheung W. Y. Cyclic 3',5'-nucleotide phosphodiesterase. Demonstration of an activator. Biochem Biophys Res Commun. 1970 Feb 6;38(3):533–538. doi: 10.1016/0006-291x(70)90747-3. [DOI] [PubMed] [Google Scholar]
  3. Cheung W. Y. Cyclic 3',5'-nucleotide phosphodiesterase: pronounced stimulation by snake venom. Biochem Biophys Res Commun. 1967 Nov 30;29(4):478–482. doi: 10.1016/0006-291x(67)90508-6. [DOI] [PubMed] [Google Scholar]
  4. Clarke R. G., Howlett G. J. Determination of the molecular weight of proteins in heterogeneous mixtures: use of an air-driven ultracentrifuge for the analysis of protein--protein interactions. Arch Biochem Biophys. 1979 Jun;195(1):235–242. doi: 10.1016/0003-9861(79)90345-x. [DOI] [PubMed] [Google Scholar]
  5. Crouch T. H., Klee C. B. Positive cooperative binding of calcium to bovine brain calmodulin. Biochemistry. 1980 Aug 5;19(16):3692–3698. doi: 10.1021/bi00557a009. [DOI] [PubMed] [Google Scholar]
  6. Dedman J. R., Potter J. D., Jackson R. L., Johnson J. D., Means A. R. Physicochemical properties of rat testis Ca2+-dependent regulator protein of cyclic nucleotide phosphodiesterase. Relationship of Ca2+-binding, conformational changes, and phosphodiesterase activity. J Biol Chem. 1977 Dec 10;252(23):8415–8422. [PubMed] [Google Scholar]
  7. Klee C. B., Crouch T. H., Richman P. G. Calmodulin. Annu Rev Biochem. 1980;49:489–515. doi: 10.1146/annurev.bi.49.070180.002421. [DOI] [PubMed] [Google Scholar]
  8. Ogawa Y. The apparent binding constant of glycoletherdiaminetetraacetic acid for calcium at neutral pH. J Biochem. 1968 Aug;64(2):255–257. doi: 10.1093/oxfordjournals.jbchem.a128887. [DOI] [PubMed] [Google Scholar]
  9. Sharma R. K., Wang J. H. Preparation and assay of the Ca2+--dependent modulator protein. Adv Cyclic Nucleotide Res. 1979;10:187–198. [PubMed] [Google Scholar]
  10. Sharma R. K., Wang T. H., Wirch E., Wang J. H. Purification and properties of bovine brain calmodulin-dependent cyclic nucleotide phosphodiesterase. J Biol Chem. 1980 Jun 25;255(12):5916–5923. [PubMed] [Google Scholar]
  11. Teo T. S., Wang J. H. Mechanism of activation of a cyclic adenosine 3':5'-monophosphate phosphodiesterase from bovine heart by calcium ions. Identification of the protein activator as a Ca2+ binding protein. J Biol Chem. 1973 Sep 10;248(17):5950–5955. [PubMed] [Google Scholar]
  12. Wang J. H., Waisman D. M. Calmodulin and its role in the second-messenger system. Curr Top Cell Regul. 1979;15:47–107. doi: 10.1016/b978-0-12-152815-7.50006-5. [DOI] [PubMed] [Google Scholar]
  13. Watterson D. M., Van Eldik L. J., Smith R. E., Vanaman T. C. Calcium-dependent regulatory protein of cyclic nucleotide metabolism in normal and transformed chicken embryo fibroblasts. Proc Natl Acad Sci U S A. 1976 Aug;73(8):2711–2715. doi: 10.1073/pnas.73.8.2711. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Wolff D. J., Brostrom C. O. Calcium-binding phosphoprotein from pig brain: identification as a calcium-dependent regulator of brain cyclic nucleotide phosphodiesterase. Arch Biochem Biophys. 1974 Jul;163(1):349–358. doi: 10.1016/0003-9861(74)90486-x. [DOI] [PubMed] [Google Scholar]
  15. Yazawa M., Yagi K. Purification of modulator-deficient myosin light-chain kinase by modulator protein-Sepharose affinity chromatography. J Biochem. 1978 Nov;84(5):1259–1265. doi: 10.1093/oxfordjournals.jbchem.a132244. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES