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T. M. Grau, N. O. Artemyev, H. Muradov, B. Wissinger, S. Kohl; Functional Analysis of PDE6C Mutations Associated With Autosomal Recessive Achromatopsia. Invest. Ophthalmol. Vis. Sci. 2009;50(13):4793.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate the functional consequences of five missense mutations in PDE6C, the gene encoding the alpha’- subunit of the cone-specific phosphodiesterase. Mutations in PDE6C are a rare cause of autosomal recessive achromatopsia.
Expression constructs applying the pFastBac HTb vector (Invitrogen, Carlsbad, USA) for human PDE6C/PDE5-chimeras were cloned and achromatopsia associated mutations introduced by an in vitro mutagenesis strategy. Proteins were expressed in the baculovirus-Sf9-expression system and monitored by SDS-PAGE, Western blotting and Coomassie staining. The proteins were purified using affinity chromatography on a His-bind resin (Novagen, Darmstadt, Germany). The functionality of wild type and mutant recombinant proteins was analyzed by a series of assays including gel filtration, enzymatic activity, and inhibition by Zaprinast, IBMX and the inhibitory pγ-subunit.
All mutations result in considerable to total loss of PDE6C-specific enzymatic activity and/or altered substrate binding, as well as altered binding and inhibition by Zaprinast, IBMX and the inhibitory pγ-subunit.
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