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RH Cote, AE Daly, BA Valeriani, N Vardi; Regulation of Cone Photoreceptor Phosphodiesterase (PDE6C) by Its Inhibitory ’ Subunit and by cGMP Binding . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1960.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To improve existing purification strategies for bovine cone PDE6C to test the idea that differences in the photoresponse of rods and cones can be ascribed in part to differences in PDE6 regulation. Methods: Soluble PDE6 was initially isolated from bovine retinas following the approach of Gillespie and Beavo (JBC 263, 8133 (1988)). Cone PDE6C was separated from rod PDE6 by Q-Sepharose chromatography, followed by hydroxyapatite and gel filtration chromatography. Rod- and cone-specific anti-peptide antibodies to catalytic and inhibitory subunits were developed and affinity-purified to identify the different PDE6 isoforms in immunoblots and immunocytochemistry. Activity measurements and binding affinity for cGMP or γ were performed using standard procedures [Cote, Meth. Enzymol. 315, 646 (2000)]. Results: PDE6C was purified by three successive chromatographic procedures to ∼90% purity. Immunoblot analysis with rod and cone-specific PDE6 antibodies revealed no significant rod PDE6 catalytic or inhibitory subunit contamination in the purified PDE6C. PDE6C co-purifies with the 17 kDa δ subunit as well as other δ-immunoreactive proteins. Immunocytochemical analysis of bovine retinas with these antibodies demonstrated strong staining of cone γ’ and α’ subunit antibodies in both blue and red/green cone outer segments, but not in rod outer segments; a rod γ-specific antibody localized to rod–but not cone–outer segments. The catalytic properties (Km, kcat) of PDE6C were not greatly different from rod PDE6. Second-generation PDE5-specific inhibitors were 7-10-fold less effective in inhibiting activated PDE6C (E4021 Ki = 22 nM) compared to rod PDE6, whereas zaprinast and IBMX were much less potent and discriminated cone and rod PDE6 less well. Analysis of both cGMP binding to GAF domains and γ' binding to the catalytic dimer reveal binding heterogeneity similar to rod PDE6, but in both cases the affinity of these interaction are weaker for PDE6C. Conclusions: Cone-specific isoforms of PDE6 catalytic and inhibitory subunits are expressed in both red/green and blue cone photoreceptors in bovine retina. Observed differences in the allosteric regulation and/or pharmacological properties of PDE6C will be relevant to understanding how rod and cone photoreceptors respond differently to illumination or to therapeutic drugs.
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