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V. A. Bondarenko, A. Yamazaki; Activation of Mammalian Retinal cGMP Phosphodiesterase Requires Dissociation of Its Inhibitory Subunit. Invest. Ophthalmol. Vis. Sci. 2008;49(13):1678. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
The purpose of this study is to elucidate the molecular mechanisms of activation of PDE using purified enzymes.
We purified three different forms of retinal cGMP phosphodiesterase (PDE) from bovine outer segments of retinal photoreceptors (OS) and compared their activity and subunit composition.
After incubation of OS homogenates with GTP or guanosine 5’-O-(3-thiotriphosphate) (GTPγS), PDE species were extracted form membranes with a hypotonic buffer and isolated with an anion exchange column. Without guanine nucleotide, one PDE species, PDEαβγγ, was eluted. With guanine nucleotides, PDEαβγγ, PDEαβγ and a minute amount of a PDEαβ/Pγ complex were eluted. Isolated PDEαβγ showed a higher enzymatic activity (~12 times) and a higher Pγ sensitivity (~30 times) than those of PDEαβγγ. It is important to note that the enzymatic activity of PDEαβγ emerged in the presence of cGMP was much higher (~24 times), implying that Pαβγ isolated is a mixture of at least two Pαβγ forms and that the PDEαβγ form having a higher PDE activity is emerged at the initial stage of phototransduction. Pαβγγ and Pαβγ were also proportionally extracted with PDEΔ in an isotonic buffer although its numbers bound to these PDE species are different, as PDEαβγγΔ and PDEαβγΔΔ. However, neither PDEαβ nor the PDEαβγ complex with GTPγS-Tα was extracted under any conditions. We also found that cone PDE is activated in a manner similar to that of rod PDE.
We propose that complete dissociation of one Pγ from PDEαβγγ by GTP-Tα is absolutely necessary for PDE activation and that PDEαβγ is its activated form. The basic mechanism for PDE activation is very similar in mammalian and amphibian OS and also in rods and cones.
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