Abstract
Size-exclusion chromatography (gel filtration) is a widely used method to determine the molecular weight of a protein. Often, the elution volume of several standard proteins is plotted against their known molecular weight to generate a standard curve, which is then used to determine the molecular weight of the protein of interest by its elution volume. However, gel filtration does not measure the mass of a particle as such, but the Stokes radius (R s), a property dependent on mass, shape, and hydration of a protein. Thus, this method works well only if the protein of interest has a spherical symmetrical shape and an average hydration level. For all other proteins, the use of gel filtration as the sole means to determine the molecular weight will be misleading. The molecular weight of any given protein can be calculated, however, using the method of Siegel and Monty. This method combines Stokes radii obtained from gel filtrations and sedimentation coefficients derived from density gradient centrifugations to calculate the mass of a protein independently of its shape or hydration. It has been shown previously that PDE4D3, a representative of the long PDE4 splice forms, behaves as a dimer, whereas PDE4D2, a prototype of the short PDE4 splice forms, is a monomer. Both proteins exhibit an anomalous behavior on gel filtration columns. For this reason, they are used in this study to demonstrate the necessity of performing both gel filtration and density gradient centrifugation to determine the molecular weight of a protein.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Francis, S. H., Turko, I. V., and Corbin, J. D. (2001) Cyclic nucleotide phosphodiesterases: relating structure and function. Prog. Nucleic Acid Res. Mol. Biol. 65, 1–52.
Kakkar, R., Raju, R. V., and Sharma, R. K. (1999) Calmodulin-dependent cyclic nucleotide phosphodiesterase (PDE1). Cell. Mol. Life Sci. 55, 1164–1186.
Francis, S. H., Chu, D. M., Thomas, M. K., et al. (1998) Ligand-induced conformational changes in cyclic nucleotide phosphodiesterases and cyclic nucleotide-dependent protein kinases. Methods 14, 81–92.
Grange, M., Sette, C., Cuomo, M., Conti, M., Lagarde, M., Prigent, A. F., and Nemoz, G. (2000) The cAMP-specific phosphodiesterase PDE4D3 is regulated by phosphatidic acid binding: consequences for cAMP signaling pathway and characterization of a phosphatidic acid binding site. J. Biol. Chem. 275, 33,379–33,387.
Sharma, R. K. and Wang, J. H. (1986) Calmodulin and Ca2+-dependent phosphorylation and dephosphorylation of 63-kDa subunit-containing bovine brain calmodulin-stimulated cyclic nucleotide phosphodiesterase isozyme. J. Biol. Chem. 261, 1322–1328.
MacKenzie, S. J., Baillie, G. S., McPhee, I., et al. (2002) Long PDE4 cAMP specific phosphodiesterases are activated by protein kinase A-mediated phosphorylation of a single serine residue in Upstream Conserved Region 1 (UCR1). Br. J. Pharmacol. 136, 421–433.
Sette, C. and Conti, M. (1996) Phosphorylation and activation of a cAMP-specific phosphodiesterase by the cAMP-dependent protein kinase: involvement of serine 54 in the enzyme activation. J. Biol. Chem. 271, 16,526–16,534.
Ahmad, F., Cong, L. N., Stenson Holst, L., Wang, L. M., Rahn Landstrom, T., Pierce, J. H., Quon, M. J., Degerman, E., and Manganiello, V. C. (2000) Cyclic nucleotide phosphodiesterase 3B is a downstream target of protein kinase B and may be involved in regulation of effects of protein kinase B on thymidine incorporation in FDCP2 cells. J. Immunol. 164, 4678–4688.
Thomas, M. K., Francis, S. H., and Corbin, J. D. (1990) Substrate-and kinase-directed regulation of phosphorylation of a cGMP-binding phosphodiesterase by cGMP. J. Biol. Chem. 265, 14,971–14,978.
Stroop, S. D. and Beavo, J. A. (1991) Structure and function studies of the cGMP-stimulated phosphodiesterase. J. Biol. Chem. 266, 23,802–23,809.
Martinez, S. E., Wu, A. Y., Glavas, N. A., Tang, X. B., Turley, S., Hol, W. G., and Beavo, J. A. (2002) The two GAF domains in phosphodiesterase 2A have distinct roles in dimerization and in cGMP binding. Proc. Natl. Acad. Sci. USA 99, 13,260–13,265.
Fink, T. L., Francis, S. H., Beasley, A., Grimes, K. A., and Corbin, J. D. (1999) Expression of an active, monomeric catalytic domain of the cGMP-binding cGMP-specific phosphodiesterase (PDE5). J. Biol. Chem. 274, 34,613–34,620.
Kameni Tcheudji, J. F., Lebeau, L., Virmaux, N., Maftei, C. G., Cote, R. H., Lugnier, C., and Schultz, P. (2001) Molecular organization of bovine rod cGMP-phosphodiesterase 6. J. Mol. Biol. 310, 781–791.
Muradov, K. G., Boyd, K. K., Martinez, S. E., Beavo, J. A., and Artemyev, N. O. (2003) The GAFa domains of rod cGMP-phosphodiesterase 6 determine the selectivity of the enzyme dimerization. J. Biol. Chem. 278, 10,594–10,601.
Richter, W. and Conti, M. (2002) Dimerization of the type 4 cAMP-specific phosphodiesterases is mediated by the upstream conserved regions (UCRs). J. Biol. Chem. 277, 40,212–40,221.
Kovala, T., Sanwal, B. D., and Ball, E. H. (1997) Recombinant expression of a type IV, cAMP-specific phosphodiesterase: characterization and structure-function studies of deletion mutants. Biochemistry 36, 2968–2976.
Rocque, W. J., Holmes, W. D., Patel, I. R., Dougherty, R. W., Ittoop, O., Overton, L., Hoffman, C. R., Wisely, G. B., Willard, D. H., and Luther, M. A. (1997) Detailed characterization of a purified type 4 phosphodiesterase, HSPDE4B2B: differentiation of high-and low-affinity (R)-rolipram binding. Protein Expr. Purif. 9, 191–202.
Richter, W., Hermsdorf, T., Lilie, H., Egerland, U., Rudolph, R., Kronbach, T., and Dettmer, D. (2000) Refolding, purification, and characterization of human recombinant PDE4A constructs expressed in Escherichia coli. Protein Expr. Purif. 19, 375–383.
Grange, M., Picq, M., Prigent, A. F., Lagarde, M., and Nemoz, G. (1998) Regulation of PDE-4 cAMP phosphodiesterases by phosphatidic acid. Cell Biochem. Biophys. 29, 1–17.
Lario, P. I., Bobechko, B., Bateman, K., Kelly, J., Vrielink, A., and Huang, Z. (2001) Purification and characterization of the human PDE4A catalytic domain (PDE4A330-723) expressed in Sf9 cells. Arch. Biochem. Biophys. 394, 54–60.
Saldou, N., Baecker, P. A., Li, B., Yuan, Z., Obernolte, R., Ratzliff, J., Osen, E., Jarnagin, K., and Shelton, E. R. (1998) Purification and physical characterization of cloned human cAMP phosphodiesterases PDE-4D and-4C. Cell Biochem. Biophys. 28, 187–217.
Xu, R. X., Hassell, A. M., Vanderwall, D., et al. (2000) Atomic structure of PDE4: insights into phosphodiesterase mechanism and specificity. Science 288, 1822–1825.
Lee, M. E., Markowitz, J., Lee, J. O., and Lee, H. (2002) Crystal structure of phosphodiesterase 4D and inhibitor complex(1). FEBS Lett. 530, 53–58.
Siegel, L. M. and Monty, K. J. (1966) Determination of molecular weights and frictional ratios of proteins in impure systems by use of gel filtration and density gradient centrifugation: application to crude preparations of sulfite and hydroxylamine reductases. Biochim. Biophys. Acta 112, 346–362.
Acknowledgments
We are indebted to Marco Conti for critical reading of the manuscript and Caren Spencer for editorial assistance. This work was supported by National Institutes of Health Grant HD20788.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2005 Humana Press Inc.
About this protocol
Cite this protocol
Richter, W. (2005). Determining the Subunit Structure of Phosphodiesterases Using Gel Filtration and Sucrose Density Gradient Centrifugation. In: Lugnier, C. (eds) Phosphodiesterase Methods and Protocols. Methods In Molecular Biology™, vol 307. Humana Press. https://doi.org/10.1385/1-59259-839-0:167
Download citation
DOI: https://doi.org/10.1385/1-59259-839-0:167
Publisher Name: Humana Press
Print ISBN: 978-1-58829-314-5
Online ISBN: 978-1-59259-839-7
eBook Packages: Springer Protocols