Abstract
Of the 11 families of mammalian cyclic nucleotide phosphodiesterases (PDEs), 5 contain regulatory domains capable of binding cyclic guanosine 5′-monophosphate (cGMP). The best understood of the GAF-containing PDEs is the family of rod (PDE6R) and cone (PDE6C) photoreceptor PDEs. Binding of cGMP to the rod PDE6 catalytic dimer (αβ) allosterically regulates the affinity of the inhibitory subunits of PDE6 (γ) for the enzyme. Two nonidentical, highaffinity cGMP-binding sites exist on the nonactivated mammalian PDE6R holoenzyme (αβγγ). One of the sites does not readily exchange with free cGMP when the catalytic dimer is complexed with Pγ. On dissociation of γ from the catalytic dimer, one of the two cGMP-binding sites undergoes a transition from high to low affinity. This chapter describes techniques to quantify cGMP binding to PDE6 in order to study the regulatory significance of the GAF domains. For high-affinity cGMP binding sites on PDE6, membrane filtration is the method of choice because of its speed, simplicity, and sensitivity. However, lower-affinity cGMP-binding sites require a method that does not perturb the equilibrium between bound and free ligand. The use of ammonium sulfate solutions during filtration extends to lower-binding affinities the useful range of membrane filtration. However, a centrifugal separation technique that minimizes perturbation of the cGMP-binding equilibrium is also presented for measuring lower-affinity cGMP-binding sites. These methods are applicable to understanding the regulatory mechanisms regulating other GAF-containing PDEs as well.
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Acknowldgments
I am grateful to the past and present members of my laboratory for developing the procedures and conducting the experiments reported in this chapter. This work was supported by the National Institutes of Health (National Eye Institute, EY-05798). This is Scientific Contribution #2191 from the New Hampshire Agricultural Experiment Station.
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Cote, R.H. (2005). Cyclic Guanosine 5′-Monophosphate Binding to Regulatory GAF Domains of Photoreceptor Phosphodiesterase. In: Lugnier, C. (eds) Phosphodiesterase Methods and Protocols. Methods In Molecular Biology™, vol 307. Humana Press. https://doi.org/10.1385/1-59259-839-0:141
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DOI: https://doi.org/10.1385/1-59259-839-0:141
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