Journal: The FEBS journal

Article Title: Binding of cGMP to the transducin-activated cGMP phosphodiesterase, PDE6, initiates a large conformational change involved in its deactivation

doi: 10.1111/j.1742-4658.2011.08104.x

Figure Lengend Snippet: Binding of [3H]cGMP to membranous PDE. A. Levels of [3H]cGMP binding to OS membranes treated with or without GTPγS. OS homogenates (27.5 mg protein) were suspended in 18.4 ml of Buffer A and divided into two portions. After incubation of a portion with 50 μM GTPγS overnight on ice, its membranes were washed with 5 ml Buffer A supplemented with 50 μM GTPγS (×2) and 5 ml Buffer A (×2) and suspended in 5 ml Buffer A. The other portion was treated in the same way but without GTPγS. Binding of [3H]cGMP to these suspensions (10 μl) was assayed using 1 μM [3H]cGMP. B and C. [3H]cGMP binding to proteins extracted from OS membranes treated with or without GTPγS. OS homogenates (27.7 mg protein) were suspended in 18 ml of Buffer A, divided into two portions and treated with or without GTPγS. Proteins were extracted from membranes with 3 ml Buffer B (×7), concentrated to ∼0.5 ml and applied to Bio-Gel A-0.5 m column. [3H]cGMP-binding activity (B) and PDE activity (not shown) were assayed using 60 and 5 μl of the fraction, respectively. Protein profiles in the fraction (90 μl) were analyzed by SDS-PAGE and staining with Coomassie blue (C). The left end lane shows Mr of standard proteins, 94, 67 and 43 kDa.

Article Snippet: Contents of cGMP bound to purified Pαβγγ and Pαβγ were measured using a cGMP immunoassay kit, the Correlate-EIA Direct Cyclic GMP Enzyme Immunoassay Kit (Assay Designs, Ann Arbor, MI).

Techniques: Binding Assay, Incubation, Activity Assay, SDS Page, Staining

Journal: The FEBS journal

Article Title: Binding of cGMP to the transducin-activated cGMP phosphodiesterase, PDE6, initiates a large conformational change involved in its deactivation

doi: 10.1111/j.1742-4658.2011.08104.x

Figure Lengend Snippet: Binding of [3H]cGMP to PDE species extracted from OS membranes. A and B. PDE species extracted with a hypotonic buffer. Details of the procedure are in Experimental procedures. OS homogenates (50.4 mg protein) were suspended in 20 ml Buffer A and divided into three portions. After incubation with cGMP (A, upper panel), GTPγS (A, lower panel) or cGMP + GTPγS (B), proteins were extracted with Buffer B (a hypotonic buffer), applied to a TSK-DEAE 5PW column and eluted. Fractions containing PDE species were determined by SDS-PAGE and assaying PDE activity. Elution profiles of the 88-kDa-protein, Pαβ, are shown in each panel. Elution profile of other proteins was shown in Ref 20. PDE species were identified as described (20). Binding of [3H]cGMP to the fraction (60 μl) was measured with 0.5 μM [3H]cGMP. C. PDE species extracted with Pδ in an isotonic buffer. OS homogenates (12.4 mg) were suspended in 13 ml of Buffer A and divided into two portions. After incubation of a portion with GTPγS (50 μM) for 1 h on ice, membranes were washed with 2 ml of Buffer A containing GTPγS (50 μM) and 2 ml of Buffer A. The other portion was treated in the same way but without GTPγS. These membranes were suspended in 2.5 ml of Buffer D, incubated with Pδ (final 3 μM) overnight on ice, and washed with 2 ml of Buffer D (×2). All supernatants were collected and applied to a TSK-DEAE 5PW column. Rod and cone PDE species and their stoichiometry and transducin subunits were identified as described (20). Binding of [3H]cGMP to the fraction (50 μl) was measured with 0.5 μM [3H]cGMP (upper panel). Protein profiles in fractions (40 μl) were analyzed by SDS-PAGE and staining with Coomassie blue (lower panel). Due to the limited space, only results from GTPγS-treated membranes are shown. Profiles of PDE species from GTPγS-non-treated membranes are in Ref. 20.

Article Snippet: Contents of cGMP bound to purified Pαβγγ and Pαβγ were measured using a cGMP immunoassay kit, the Correlate-EIA Direct Cyclic GMP Enzyme Immunoassay Kit (Assay Designs, Ann Arbor, MI).

Techniques: Binding Assay, Incubation, SDS Page, Activity Assay, Staining

Journal: The FEBS journal

Article Title: Binding of cGMP to the transducin-activated cGMP phosphodiesterase, PDE6, initiates a large conformational change involved in its deactivation

doi: 10.1111/j.1742-4658.2011.08104.x

Figure Lengend Snippet: Pγ effect on the conformational change of Pαβγ by cGMP binding. A. The time to detect the increase in the level of [3H]cGMP-bound Pαβγ trapped by the filter. The experiment was carried out as a part of the study depicted in Figure 5A. Pαβγ (16.0 μg) was suspended in 640 μl of 55.5 mM Tris·HCl (pH 7.5) containing 4.44 mM EDTA and 1.11 mM IBMX, and [3H]cGMP-binding to the Pαβγ was initiated by adding 80 μl of 9 μM [3H]cGMP. After incubation for 30 min, an aliquot (72 μl) was withdrawn and applied to a Millipore filter, and its radioactivity was designated as the level at time 0. Simultaneously, a mixture (72 μl) of Pγ or its mutant (10 μM) with or without cGMP (10 mM) was added to the assay mixture: (●), with cGMP; and (○), without cGMP. After incubation for 0.25, 0.5, 0.75, 1, 2, 5, 10, and 20 min, an aliquot (80 μl) was withdrawn and applied to a Millipore filter, and its radioactivity was measured. The arrow (↑) indicates the addition of Pγ (or Pγ mutant) with or without cGMP. The arrow (⇦) indicates levels of [3H]cGMP-bound Pαβγ with (▲) or without (△) N18del. The 100% activity indicates that 1.32 pmol of [3H]cGMP was detected in 1.6 μg of Pαβγ (7.72 pmol). Data shown in Figure 5A was used as a control for this study. B. Elution profile of PDE species from a gel filtration column. Purified Pαβγ (70 μg) was incubated with unlabeled cGMP (0.5 mM) in 0.5 ml of 25 mM Tris·HCl, pH 7.5, 0.1 mM EDTA, and 1 mM IBMX for 30 min on ice and applied to a Superdex 200 HR column that had been equilibrated with Buffer E (black). The chromatography conditions are in Experimental Procedure. PDE activity (●) was assayed using 5 μl of the fraction. [3H]cGMP-binding activity (□) was measured using 50 μl of the fraction. Pαβγγ (90 μg) was also applied to the column and eluted in the same manner (red). PDE activity was assayed using 20 μl of the fraction (●). The 100% PDE activity indicates that 12.1 nmol cGMP hydrolyzed/min/tube. [3H]cGMP-binding activity was measured using 50 μl of the fraction (□). The 100% [3H]cGMP-binding activity indicates that 2.4 pmol of [3H]cGMP was detected in the assay mixture.

Article Snippet: Contents of cGMP bound to purified Pαβγγ and Pαβγ were measured using a cGMP immunoassay kit, the Correlate-EIA Direct Cyclic GMP Enzyme Immunoassay Kit (Assay Designs, Ann Arbor, MI).

Techniques: Binding Assay, Incubation, Radioactivity, Mutagenesis, Activity Assay, Control, Filtration, Purification, Chromatography

Journal: The FEBS journal

Article Title: Binding of cGMP to the transducin-activated cGMP phosphodiesterase, PDE6, initiates a large conformational change involved in its deactivation

doi: 10.1111/j.1742-4658.2011.08104.x

Figure Lengend Snippet: Change of Pαβγ's characteristics by cGMP binding. A. Dissociation of [3H]cGMP bound to Pαβγ. Purified Pαβγ (16.0 μg) suspended in 640 μl of 55.5 mM Tris·HCl (pH 7.5) containing 4.44 mM EDTA and 1.11 mM IBMX, and [3H]cGMP binding was initiated by adding 80 μl of 9 μM [3H]cGMP. After incubation for 30 min on ice, an aliquot (72 μl) was withdrawn and applied to a Millipore filter, and its radioactivity was designated as the level at time 0. Simultaneously, 72 μl of 10 mM unlabeled cGMP (●) or water (○) was added to the assay mixture. After incubation for 0.25, 0.5, 0.75, 1, 2, 5, 10, and 20 min, an aliquot (80 μl) was withdrawn and applied to a Millipore filter, and its [3H]-radioactivity was measured. The arrow indicates the addition of cGMP or water. The 100% activity indicates that 1.32 pmol of [3H]cGMP was detected in 1.6 μg of Pαβγ (7.72 pmol). B. Elution profile of Pαβγ from a gel filtration column. Purified Pαβγ (70 μg) was incubated with (black) or without (red) unlabeled cGMP (0.5 mM) in 0.5 ml of 25 mM Tris·HCl, pH 7.5, 0.1 mM EDTA, and 1 mM IBMX for 30 min on ice and applied to a Superdex 200 HR column that had been equilibrated with Buffer E. Detailed conditions for this elution are in Experimental Procedure. PDE activity was assayed using 5 μl of the fraction (●). The 100% PDE activity indicates that 12.5 nmol cGMP hydrolyzed/min/tube. [3H]cGMP binding activity was measured using 50 μl of the fraction (□). The 100% activity indicates that 1.50 pmol of [3H]cGMP was detected in the assay mixture.

Article Snippet: Contents of cGMP bound to purified Pαβγγ and Pαβγ were measured using a cGMP immunoassay kit, the Correlate-EIA Direct Cyclic GMP Enzyme Immunoassay Kit (Assay Designs, Ann Arbor, MI).

Techniques: Binding Assay, Purification, Incubation, Radioactivity, Activity Assay, Filtration

Journal: The FEBS journal

Article Title: Binding of cGMP to the transducin-activated cGMP phosphodiesterase, PDE6, initiates a large conformational change involved in its deactivation

doi: 10.1111/j.1742-4658.2011.08104.x

Figure Lengend Snippet: Levels of cGMP contained in Pαβγγ and Pαβγ. Pαβγγ (6.50 μg/20 μl) and Pαβγ (4.75 μg/50 μl) were purified from GTPγS-treated OS homogenates. A. Purity of these PDE preparations. Preparations of Pαβγγ (10 μl) and Pαβγ (25 μl) were applied to SDS-PAGE followed by staining with Coomassie blue. B. Levels of cGMP contained in these PDE species. Contents of cGMP were measured using a cGMP immunoassay kit.

Article Snippet: Contents of cGMP bound to purified Pαβγγ and Pαβγ were measured using a cGMP immunoassay kit, the Correlate-EIA Direct Cyclic GMP Enzyme Immunoassay Kit (Assay Designs, Ann Arbor, MI).

Techniques: Purification, SDS Page, Staining

Journal: The FEBS journal

Article Title: Binding of cGMP to the transducin-activated cGMP phosphodiesterase, PDE6, initiates a large conformational change involved in its deactivation

doi: 10.1111/j.1742-4658.2011.08104.x

Figure Lengend Snippet: Binding of [3H]cGMP to Pαβγ. A. Concentration of [3H]cGMP. [3H]cGMP binding to Pαβγ (1.92 μg) was measured with indicated concentrations of [3H]cGMP. The [3H]cGMP-binding activity was analyzed by Scatchard plotting (Insert). B. Time-course. Pαβγ (17.3 μg) was incubated in 55 mM Tris·HCl, (pH 7.5) containing 4.4 mM EDTA and 1.1 mM IBMX (final volume, 720 μl) on ice for 10 min. The [3H]cGMP binding was initiated by adding 80 μl of 10 μM [3H]cGMP. After incubation for indicated periods, an aliquot (80 μl) was taken and applied to a Millipore filter. C. The cyclic nucleotide-specificity. After incubation of Pαβγ (1.92 μg) with indicated concentration of unlabeled cGMP (●) or cAMP (○) on ice for 10 min, [3H]cGMP binding was measured with 1 μM [3H]cGMP. The 100% activity indicates that 1.46 pmol [3H]cGMP bound to Pαβγ in tubes. D. Levels of [3H]cGMP-bound Pαβγ trapped by the filter. OS homogenates (18.9 mg protein) were suspended in 9.7 ml of Buffer A. After isolation by the TSK-DEAE 5PW column chromatography and concentration to 0.3 ml, the Pαβγ preparation (∼80 μg) was incubated with 1 μM [3H]cGMP for 30 min on ice and applied to a TSK 250 column that had been equilibrated with Buffer D. The level of [3H]cGMP bound to Pαβγ was calculated based on the [3H]-radioactivity in 70 μl of the fraction (●). The fraction (70 μl) was also applied to a Millipore filter and the [3H]-radioactivity on the filter was measured (□). Only fractions containing Pαβγ were shown. Insert: The rate of [3H]-radioactivity on the filter per the level of [3H]-radioactivity in the fraction. The 100% radioactivity indicates the [3H]-radioactivity detected in fraction 15. Fraction 15 (70 μl) contained 3.2 μg Pαβγ (15.5 pmol) and 13.1 pmol of [3H]cGMP.

Article Snippet: Contents of cGMP bound to purified Pαβγγ and Pαβγ were measured using a cGMP immunoassay kit, the Correlate-EIA Direct Cyclic GMP Enzyme Immunoassay Kit (Assay Designs, Ann Arbor, MI).

Techniques: Binding Assay, Concentration Assay, Activity Assay, Incubation, Isolation, Column Chromatography, Radioactivity

Journal: The FEBS journal

Article Title: Binding of cGMP to the transducin-activated cGMP phosphodiesterase, PDE6, initiates a large conformational change involved in its deactivation

doi: 10.1111/j.1742-4658.2011.08104.x

Figure Lengend Snippet: Effects of Pγ and its mutants on [3H]cGMP binding to Pαβγ. A. The effect on the level of [3H]cGMP binding. After incubation or Pαβγ (1.92 μg) with various concentrations of Pγ or its mutants, the [3H]cGMP-binding activity was measured. The 100% activity indicates that 1.46 pmol [3H]cGMP bound to Pαβγ in tubes. Following Pγ and its mutants were used: ●, wild type Pγ; □, N18del; △, N22del; ▲, C18Sub, and ▼, C10del. B. The effect on the time-course of [3H]cGMP-binding. Pαβγ (17.3 μg) was incubated with 1.11 μM Pγ or its mutants in 55 mM Tris·HCl, (pH 7.5) containing 4.4 mM EDTA and 1.1 mM IBMX (final volume, 720 μl) on ice for 30 min. The [3H]cGMP binding was initiated by adding 80 μl of 10 μM [3H]cGMP. After incubation for indicated periods on ice, an aliquot (80 μl) was taken and applied to a Millipore filter. Following Pγ and its mutants were used: ○, control; ●, wild type Pγ; △, N22del; and ▲, C18Sub. C. The effect on the Scatchard plot. Pαβγ (1.92 μg) was incubated with 1 mM of wild type Pγ (●) or N22del (△). As a control, Pαβγ alone was incubated (○). Then, [3H]cGMP binding was initiated by adding indicated concentrations of [3H]cGMP (C-1). The [3H]cGMP binding in C-1 is analyzed by Scatchard plotting (C-2).

Article Snippet: Contents of cGMP bound to purified Pαβγγ and Pαβγ were measured using a cGMP immunoassay kit, the Correlate-EIA Direct Cyclic GMP Enzyme Immunoassay Kit (Assay Designs, Ann Arbor, MI).

Techniques: Binding Assay, Incubation, Activity Assay, Control

Journal: The FEBS journal

Article Title: Binding of cGMP to the transducin-activated cGMP phosphodiesterase, PDE6, initiates a large conformational change involved in its deactivation

doi: 10.1111/j.1742-4658.2011.08104.x

Figure Lengend Snippet: Role of the non-catalytic cGMP-binding site in PDE regulation. Activation. GTP-Tα activates Pαβγγ/2cGMPs (Pαβγγ having two cGMPs) to Pαβγ/cGMP (Pαβγ having one cGMP). At the initial stage, cGMP is present in OS; however, binding of cGMP to the empty site on Pαβγ/cGMP is suppressed. Deactivation. After hydrolysis of cGMP, retinal guanylate cyclase initiates to produce cGMP from GTP. When the [cGMP] in OS is increased to ∼50 nM, the cGMP binds to Pαβγ/cGMP and Pαβγ/2cGMPs is formed. Then, the PDE species slowly changes its conformation. Interaction with Pγ·GDP-Tα accelerates the conformational change and swiftly establishes the inactive form of PDE (Pαβγγ/2cGMPs).

Article Snippet: Contents of cGMP bound to purified Pαβγγ and Pαβγ were measured using a cGMP immunoassay kit, the Correlate-EIA Direct Cyclic GMP Enzyme Immunoassay Kit (Assay Designs, Ann Arbor, MI).

Techniques: Binding Assay, Activation Assay

Journal: Frontiers in Physiology

Article Title: Combined Systemic Intake of K-ATP Opener (Nicorandil) and Mesenchymal Stem Cells Preconditioned With Nicorandil Alleviates Pancreatic Insufficiency in a Model of Bilateral Renal Ischemia/Reperfusion Injury

doi: 10.3389/fphys.2022.934597

Figure Lengend Snippet: Estimated levels of blood glucose, LDH, amylase, MPO, and TNF-α in all studied groups ( n = 6 in each group). p -value ≤0.05 is considered significant.

Article Snippet: Then using quantitative Sandwich Rat Cyclic Guanosine Monophosphate (c-GMP) ELISA Kit (MyBioSource, Catalog #MBS007871, the level of c-GMP was estimated and expressed per ml.

Techniques: