PDE1A Assay Kit

• Background:

  Phosphodiesterases (PDEs) play an important role in the dynamic regulation of the second messengers cAMP (cyclic adenosine monophosphate) and cGMP (cyclic guanosine monophosphate) signaling, by hydrolyzing them. The PDE superfamily is composed of 11 families, with PDE4,7 and 8 being cAMP-specific hydrolases, and thus regulating positive and negative responses to it. Among the 11 different families of PDEs, phosphodiesterase 1 (PDE1) sub-family of enzymes hydrolyze both cAMP and cGMP in a mutually competitive manner. The catalytic activity of PDE1 is stimulated by their binding to Ca2+/calmodulin, resulting in the integration of Ca2+ and cyclic nucleotide-mediated signaling in various diseases. The PDE1 family includes three subtypes, PDE1A, PDE1B and PDE1C, which differ for their relative affinities for cAMP and cGMP. These isoforms are differentially expressed throughout the body, including the cardiovascular, central nervous system and other organs. PDE1A increases vascular contraction of mesenteric arteries upon angiotensin II infusion in rats and also regulates cGMP hydrolysis in cardiomyocyte and vascular smooth muscle cells, which in turn controls smooth muscle tone. Inhibition of PDE1A attenuates adverse cardiac remodeling and may serve as a novel therapeutic approach to limit nitrate tolerance.

• Description:

  The PDE1A Assay Kit is a fluorescence polarization (FP), homogeneous, 96-well assay kit designed for the screening and profiling of PDE1A (Phosphodiesterase 1A) inhibitors. This assay takes advantage of a specific fluorescent phosphate-binding nanoparticle. The kit contains enough purified recombinant PDE1A, fluorescent probe, PDE assay buffer, Binding Agent, and diluent for 100 reactions.Figure 1: Illustration of the PDE1A Assay Kit principle.The assay uses a fluorescein-labeled cyclic adenosine monophosphate (cAMP-FAM for PDE1A), in which the phosphate group is engaged within the cyclic nucleotide. This is a very small molecule that rotates fast (low FP) . PDE1A catalyzes the hydrolysis of the phosphodiester bond in the cyclic nucleotide and frees the phosphate group. In a second step the free phosphate group is recognized by a specific phosphate-binding nanobead (Binding Agent) leading to the formation of a large complex, with restricted movement (high FP) . FP is proportional to PDE1A activity.This assay requires a fluorescent microplate reader capable of measuring fluorescence polarization (FP) and equipped with the required parts to read the FP signal. For more information FP technology, visit our Tech Note:FP, assay principles and applications.Note:As of August 2024, this protocol has been re-optimized for performance. Previous versions of this kit are available upon request.Need us to run inhibitor screens or profile your compounds against PDE1A? Check out ourPhosphodiesterase Screening Services.

• UniProt:

  P54750-4

• Applications:

  Study enzyme kinetics and screen small molecule inhibitors for drug discovery in high throughput screening (HTS) applications.

• Format:

  Catalog #NameAmountStorage60010PDE1A, GST-tag*˃1 µg-80°C60200FAM-Cyclic-3′, 5′-AMP**1.2 nmoles**-80°C60393PDE Assay Buffer (Incomplete) 25 ml-20°C60390PDE Binding Agent100 µl4°C60391PDE Binding Agent Diluent (cAMP) 10 ml4°C827350.5 M DTT200 µl-20°C79685Low binding, black 96-well plate1Room Temp.*The concentration of proteins is lot-specific and will be indicated on the tube containing the protein.**FAM-Cyclic-3′, 5′-AMP is provided as a powder. Vial will need to be resuspended in 600 µl of Complete PDE Assay Buffer before use.

• Shipping Conditions:

  -80°C

• Storage Conditions:

  This assay kit will perform optimally for up to6 monthsfrom date of receipt when the materials are stored as directed.

• Contraindications:

  The final concentration of DMSO in the assay should not exceed 1%.Fluorescent compounds that have λex=470 nm and detection at λem=528 nm can interfere with the readouts.It is recommended that the compound alone is tested to determine any potential interference of the compound on the assay results.