Interaction of PDE6β1-313 with the polycationic region of Pγ, Pγ24-45.
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A. Emission spectra of CysBC-Pγ24-45 (50 nM) alone (1) and in the presence of PDE6β1-313 (180 nM) (2) were recorded with with excitation at 445 nm. Inset: Coomassie Blue-stained gel with the sample of PDE6β1-313. B. Fluorescence of CysBC-Pγ24-45 (50 nM) was measured on addition of increasing concentrations of PDE6β1-313 (excitation at 445 nm, emission at 490 nm). The data were fit to the hyperbola equation using GraphPad Prizm software. C. Noncatalytic cGMP binding to PDE6β1-313. Binding of cGMP was carried out using [H3]cGMP and varying concentrations of unlabeled cGMP. The bound cGMP was determined by the filter-binding assay, and is expressed as a percentage of maximal cGMP binding (0.5 mol cGMP/mol PDE6β1-313). Results from one of three similar experiments are shown. The data were fit to equation for binding with ligand depletion using GraphPad Prizm software. The calculated Kd = 0.82±0.15 µM.