Purification of PDE6 Isozymes From Mammalian Retina
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 WordsPhotoreceptor phosphodiesterase retina phototransduction anion-exchange chromatography cone and rod hydrophobic interaction chromatography
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.
- 3.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.Google Scholar
- 9.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.PubMedCrossRefGoogle Scholar