Abstract
The photoreceptor phosphodiesterase (PDE6) is the central effector of visual transduction in vertebrate retinal photoreceptors. Distinct isozymes of PDE6 exist in rods and cones. Mammalian retina serves as an abundant source of tissue for PDE6 purification. Methods are described for the isolation and purification of membrane-associated PDE6 from rod outer segment membranes. Purification of cone PDE6 from the soluble fraction of retinal extracts is also described. Several procedures that can purify the rod and cone isozymes to homogeneity, including anion exchange, hydrophobic interaction, gel filtration, hydroxyapatite, and immunoaffinity chromatography, are presented. A method to activate PDE6 by limited proteolysis of its inhibitory γ-subunit is also provided.
Key Words
- Photoreceptor
- phosphodiesterase
- retina
- phototransduction
- anion-exchange chromatography
- cone and rod
- hydrophobic interaction chromatography
This is a preview of subscription content, access via your institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Cote, R. H. (2003) Structure, function, and regulation of photoreceptor phosphodiesterase (PDE6), in Handbook of Cell Signaling (Bradshaw, R. and Dennis, E., eds.), Academic, San Diego, pp. 453–457.
Hurwitz, R. L., Bunt Milam, A. H., and Beavo, J. A. (1984) Immunologic characterization of the photoreceptor outer segment cyclic GMP phosphodiesterase. J. Biol. Chem. 259, 8612–8618.
McDowell, J. H. (1993) Preparing rod outer segment membranes, regenerating rhodopsin, and determining rhodopsin concentration, in Methods in Neuroscience, vol. 15 (Hargrave, P. A., ed.), Academic, San Diego, pp. 123–130.
Kuhn, H. (1982) Light-regulated binding of proteins to photoreceptor membranes and its use for purification of several rod cell proteins. Methods Enzymol. 81, 556–564.
Gillespie, P. G. and Beavo, J. A. (1988) Characterization of a bovine cone photoreceptor phosphodiesterase purified by cyclic GMP-Sepharose chromatography. J. Biol. Chem. 263, 8133–8141.
Hurley, J. B. and Stryer, L. (1982) Purification and characterization of the gamma regulatory subunit of the cyclic GMP phosphodiesterase from retinal rod outer segments. J. Biol. Chem. 257, 11,094–11,099.
Baehr, W., Devlin, M. J., and Applebury, M. L. (1979) Isolation and characterization of cGMP phosphodiesterase from bovine rod outer segments. J. Biol. Chem. 254, 11,669–11,677.
Cote, R. H. (2000) Kinetics and regulation of cGMP binding to noncatalytic binding sites on photoreceptor phosphodiesterase. Methods Enzymol. 315, 646–672.
Körschen, H. G., Beyermann, M., Müller, F., Heck, M., Vantler, M., Koch, K. W., Kellner, R., Wolfrum, U., Bode, C., Hofmann, K. P., and Kaupp, U. B. (1999) Interaction of glutamic-acid-rich proteins with the cGMP signalling pathway in rod photoreceptors. Nature 400, 761–766.
Malinski, J. A. and Wensel, T. G. (1992) Membrane stimulation of cGMP phosphodiesterase activation by transducin: comparison of phospholipid bilayers to rod outer segment membranes. Biochemistry 31, 9502–9512.
Wensel, T. G. and Stryer, L. (1986) Reciprocal control of retinal rod cyclic GMP phosphodiesterase by its gamma subunit and transducin. Protein Struct. Funct. Genet. 1, 90–99.
Catty, P. and Deterre, P. (1991) Activation and solubilization of the retinal cGMPspecific phosphodiesterase by limited proteolysis—role of the C-terminal domain of the β-subunit. Eur. J. Biochem. 199, 263–269.
Mou, H., Grazio, H. J., Cook, T. A., Beavo, J. A., and Cote, R. H. (1999) cGMP binding to noncatalytic sites on mammalian rod photoreceptor phosphodiesterase is regulated by binding of its γ and δ subunits. J. Biol. Chem. 274, 18,813–18,820.
Artemyev, N. O. and Hamm, H. E. (1992) Two-site high-affinity interaction between inhibitory and catalytic subunits of rod cyclic GMP phosphodiesterase. Biochem. J. 283, 273–279.
Mou, H. and Cote, R. H. (2001) The catalytic and GAF domains of the rod cGMP phosphodiesterase (PDE6) heterodimer are regulated by distinct regions of its inhibitory γ subunit. J. Biol. Chem. 276, 27,527–27,534.
Bownds, D., Gordon-Walker, A., Gaide Huguenin, A. C., and Robinson, W. (1971) Characterization and analysis of frog photoreceptor membranes. J. Gen. Physiol. 58, 225–237.
Acknowledgment
This work was supported by National Eye Institute (National Institues of Health) grant EY 05798 and is Scientific Contribution Number 2195 from the New Hampshire Agricultural Experiment Station.
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
Pentia, D.C., Hosier, S., Collupy, R.A., Valeriani, B.A., Cote, R.H. (2005). Purification of PDE6 Isozymes From Mammalian Retina. In: Lugnier, C. (eds) Phosphodiesterase Methods and Protocols. Methods In Molecular Biology™, vol 307. Humana Press. https://doi.org/10.1385/1-59259-839-0:125
Download citation
DOI: https://doi.org/10.1385/1-59259-839-0:125
Publisher Name: Humana Press
Print ISBN: 978-1-58829-314-5
Online ISBN: 978-1-59259-839-7
eBook Packages: Springer Protocols