glosensor cgmp stock solution  (Promega)

Journal: The Journal of Biological Chemistry

Article Title: A unique choanoflagellate enzyme rhodopsin exhibits light-dependent cyclic nucleotide phosphodiesterase activity

doi: 10.1074/jbc.M117.775569

Figure Lengend Snippet: Enzymatic activity of Rh-PDE in HEK293 cells. A, luminescence signals representing cGMP levels, as observed in HEK293 cells with the empty vector (top), Rh-PDE (middle), and the PDE-inactive D605A mutant (bottom). HEK293 cells were preincubated in the culture medium with all-trans-retinal in the dark. The arrows and vertical light-blue line indicate sodium nitroprusside treatments and irradiation at 510 nm with 2.28 μW mm−2 intensity, respectively, and the luminescence signals of non-irradiated cells are shown as a control (black lines). B, luminescence signals representing cAMP level, as observed in HEK293 cells with the empty vector (top), Rh-PDE (middle), and the PDE-inactive D605A mutant (bottom). HEK293 cells were preincubated in the culture medium with all-trans-retinal in the dark. The arrows and vertical light-blue line indicate forskolin treatments and irradiation at 510 nm with 2.28 μW mm−2 intensity, respectively, and the luminescence signals of non-irradiated cells are shown as a control (black lines). Only HEK293 cells expressing Rh-PDE (red line in the middle panel) exhibited a decrease of the luminescence signal, indicating that cAMP concentration is lowered by light. C, changes of luminescence signals upon 2-min 510-nm irradiation (light-blue line) of HEK293 cells with the empty vector (top), Rh-PDE (middle), the PDE-inactive D605A mutant (bottom), expanded from B. Colored and black lines represent the data with and without irradiation, respectively. D, intensity dependence of the light-induced cAMP concentration decrease by Rh-PDE (cells were irradiated for 2 min with 510-nm light with 0–2.28 μW mm−2 intensities). E, peak amplitudes of luminescence decrease plotted against the light intensity (n ≧ 5 cells). Error bars, S.D. The half-maximal light intensity (EC50) was determined to be 0.244 μW mm−2 by a single exponential decay fit (red dashed line).

Article Snippet: Before measurements, the culture medium was replaced with a CO 2 -independent medium containing 10% (v/v) FBS and 2% (v/v) GloSensor cAMP or cGMP stock solution (Promega).

Techniques: Activity Assay, Plasmid Preparation, Mutagenesis, Irradiation, Expressing, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: A unique choanoflagellate enzyme rhodopsin exhibits light-dependent cyclic nucleotide phosphodiesterase activity

doi: 10.1074/jbc.M117.775569

Figure Lengend Snippet: HPLC analysis of the enzymatic activity of Rh-PDE. A, HEK293 cell membranes expressing Rh-PDE were incubated with 100 μm cGMP for 2 min (blue), 5 min (light blue), 10 min (green), 15 min (orange), and 20 min (red) in the dark (top) and under the illumination (bottom), and the reaction products were analyzed by HPLC. Dotted black line, profile for 100 μm cGMP without Rh-PDE. B, time course of the decrease of cGMP concentration in the dark (black) and under illumination (blue) (n = 3). C, cGMP hydrolysis activity in the dark and under the illumination calculated from B. D, HEK293 cell membranes expressing Rh-PDE were incubated with 100 μm cAMP for 2 min (purple), 5 min (light blue), 10 min (green), and 20 min (red) in the dark (top) and under illumination (bottom), and the reaction products were analyzed by HPLC. Dotted black line, profile for 100 μm cAMP without Rh-PDE. E, time course of the decrease of cAMP concentration in the dark (black) and under illumination (red) (n = 3). F, cAMP hydrolysis activity in the dark and under illumination calculated from E. Error bars, S.D.

Article Snippet: Before measurements, the culture medium was replaced with a CO 2 -independent medium containing 10% (v/v) FBS and 2% (v/v) GloSensor cAMP or cGMP stock solution (Promega).

Techniques: Activity Assay, Expressing, Incubation, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: A unique choanoflagellate enzyme rhodopsin exhibits light-dependent cyclic nucleotide phosphodiesterase activity

doi: 10.1074/jbc.M117.775569

Figure Lengend Snippet: Schematic representation of the Rh-PDE activation mechanism. The model shows a putative Rh-PDE dimer. A, in the dark, Rh-PDE exhibits constitutive activity toward cGMP and cAMP, suggesting that the catalytic region is somewhat exposed to the cytoplasm in mammalian cells. B, upon absorption of blue-green light by the Rh domain, photoisomerization of the retinal chromophore from all-trans-retinal (ATR) to the 13-cis form produces the M intermediate with deprotonated Schiff base (Rh domain switch on), but the PDE domain has not changed yet (PDE domain switch off). C, next, the M intermediate decays to the parent spectral state in 7 s (Rh domain switch off), accompanied by the structural changes in the PDE domain (PDE domain switch on). The resulting increased enzymatic activity is turned off within ∼70 s (PDE domain switch off).

Article Snippet: Before measurements, the culture medium was replaced with a CO 2 -independent medium containing 10% (v/v) FBS and 2% (v/v) GloSensor cAMP or cGMP stock solution (Promega).

Techniques: Activation Assay, Activity Assay