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Cyclic nucleotide phosphodiesterase assay kit

Manufactured by Enzo Life Sciences
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The Cyclic Nucleotide Phosphodiesterase Assay Kit is designed to measure the activity of cyclic nucleotide phosphodiesterase enzymes. The kit provides a colorimetric method for the determination of cyclic AMP or cyclic GMP hydrolysis.

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Lab products found in correlation

Pyrophosphate assay kit, by Abcam (1 mentions) Isopropyl β d 1 thiogalactopyranoside (iptg), by Merck Group (1 mentions) Ni nta fast start kit, by Qiagen (1 mentions) Spectramax pro, by Molecular Devices (1 mentions) Filtermax f5, by Molecular Devices (1 mentions) Pde3b, by BPS Biosciences (1 mentions)

4 protocols using cyclic nucleotide phosphodiesterase assay kit

1

Purification and Characterization of Pn Protein

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The pn CDS was cloned into the pETDuet-1vector and expressed in the Rosetta™ 2(DE3) Singles™ Competent Cells (Merck Millipore) induced with 1 mM IPTG (Merck Millipore) at 16°C overnight. The Ni-NTA Fast Start kit (Qiagen) was used to purify the N-terminal His-tagged Pn protein. After dialysis (10 mM Tris–HCl, pH 8.0), the protein aliquot was saved for future use. Pyrophosphate activity and cyclic nucleotide phosphodiesterase activity were determined using a Pyrophosphate Assay Kit (Abcam) and a Cyclic Nucleotide Phosphodiesterase Assay kit (Enzo Life Sciences). A total of 20 mM NaF was used as the pyrophosphate inhibitor, and 40 μM IBMX was used as the PDE inhibitor.
Zhang F., Qi Y., Zhou K., Zhang G., Linask K, & Xu H. (2015). The cAMP phosphodiesterase Prune localizes to the mitochondrial matrix and promotes mtDNA replication by stabilizing TFAM. EMBO Reports, 16(4), 520-527.
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2

Cyclic Nucleotide Phosphodiesterase Assay

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Experiments were conducted using 96-well plastic culture plates with cAMP or cGMP as a substrate, and PDE activity was measured using a cyclic nucleotide phosphodiesterase assay kit (Enzo Life Sciences, NY, USA). Substrates were added to the brain-derived PDE and 5′-nucleotidase, and the samples were incubated for 30 min at 30 °C separately as follows: a control group with no additive, an IBMX group (positive control for PDE inhibition), a 1 μMTH group, a 2 μMTH group, a 5 μMTH group, and a 10 μMTH group. Thereafter, BIOMOL GREEN was added, and the mixtures were stirred gently and incubated at room temperature for 20 min. Finally, the absorbance was measured at 620 nm using a microplate reader (Spectra Max Pro, Molecular Devices, Tokyo, Japan).
Amano H., Iwaki F., Oki M., Aoki K, & Ohba S. (2019). An osteogenic helioxanthin derivative suppresses the formation of bone-resorbing osteoclasts. Regenerative Therapy, 11, 290-296.
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3

Quantification of Phosphodiesterase Activity

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Caudal epididymal sperm cells (3 × 107/set suspended in 200 μl of HB buffer supplemented by 10 mM Benzamidine HCl, 0.1% 2-mercaptoethanol, 1 mM PMSF) from WT and GSK3-double hetero mice were isolated. Cells were placed in microfuge tubes in ice and sonicated (QSonica) for a total of 50 s with 10 s pulse at 40 amplitude. The sonicated cell suspensions were centrifuged at 150 00g for 15 min and supernatants were obtained. These extracts were analyzed for phosphodiesterase assay using Cyclic Nucleotide Phosphodiesterase Assay Kit, Enzo Life Sciences (BML-AK800–0001). Free phosphate ions were removed from the extract using a desalting column (BML-KI100) provided by the kit. Extracts containing PDE, substrate and 5′-nucleotidase were dissolved in assay buffer (BML-KI181) in presence or absence of IBMX and RS25344. It was incubated at 30 °C for 30 min. A parallel set for standards were prepared using 5′AMP. After incubation, reactions were terminated by addition of BIOMOL® GREEN Reagent. Color was allowed to develop for 20–30 min and absorbance at 620 nm was measured on FilterMax F5 microtiter-plate reader (Molecular Devices).
Dey S., Goswami S., Eisa A., Bhattacharjee R., Brothag C., Kline D, & Vijayaraghavan S. (2018). Cyclic AMP and glycogen synthase kinase 3 form a regulatory loop in spermatozoa. Journal of cellular physiology, 233(9), 7239-7252.
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4

Inhibitory Effect of LPE P-18:0 on PDE

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To examine the inhibitory effect of LPE P-18:0 on cyclic nucleotide PDE, Cyclic Nucleotide Phosphodiesterase Assay Kit (Enzo, cat#BML-AK800-0001) was applied following the protocol provided by the manufacturer. Briefly, the mixtures of cAMP substrate (200 nM), assay buffer, 5'-nucleotidase (1 kU/μL), and PDE3B (40 pg/reaction, BPS Bioscience, cat#60031) with LPE P-18:0 (10 μM) or IBMX (40 μM) were incubated in a microtiter plate for 0, 45, or 90 min at 30 °C. Incubation time was determined by a linearity test indicating the initial rate of the enzyme. Next, BIOMOL Green reagent was added and incubated for 30 min at 30 °C to terminate reactions. The plate was measured at 620 nm and the measure absorbance was converted to the nmol of 5'-AMP produced by using the equation for best-fit line obtained from the standard curve.
Cho Y.K., Yoon Y.C., Im H., Son Y., Kim M., Saha A., Choi C., Lee J., Lee S., Kim J.H., Kang Y.P., Jung Y.S., Ha H.K., Seong J.K., Granneman J.G., Kwon S.W, & Lee Y.H. (2022). Adipocyte lysoplasmalogenase TMEM86A regulates plasmalogen homeostasis and protein kinase A-dependent energy metabolism. Nature Communications, 13, 4084.
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