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