Adenosine deaminase ada

Manufactured by Roche

Sourced in Germany

Adenosine Deaminase (ADA) is a lab equipment product that catalyzes the deamination of adenosine to inosine. It is an important enzyme involved in the purine nucleoside metabolism pathway.

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Adenosine deaminase (11 citations)

8 protocols using adenosine deaminase ada

Adipocyte Lipolysis Measurement Assay

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Lipolysis was measured as described by Dole et al.⁴⁹ (link) Aliquots (300 μl) of a 5% suspension of adipocytes were prepared in Krebs Ringers HEPES (KRH) containing 25 mM HEPES (pH 7.4), 120 mM NaCl, 4.7 mM KCl, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄, 2.5 mM CaCl₂, 1% BSA, 2 mM glucose, and 200 nM adenosine and incubated with or without 30 nM DL-Isoproterenol hydrochloride (ISO) (Sigma-Aldrich, St. Louis, MO, USA), 10–30 U/ml adenosine deaminase (ADA) (Roche Diagnostics, Mannheim, Germany), 10 μM OPC3911 (PDE3 inhibitor) (Osaka Inc., Japan) in the presence or absence of 3 and 10 mM propionic acid (Sigma-Aldrich) or butyric acid (Sigma-Aldrich) at 37°C for 30 min in a shaking device (150 rpm). The cell-free medium was transferred to wells and buffer containing 50 mM glycine, 0.05% hydrazinehydrate, 1 mM MgCl₂, 0.75 mg/ml ATP (Sigma-Aldrich), 0.375 mg/ml NAD (Roche, Pleasanton, CA, USA), 25 μg/ml glycerol-3-phosphate dehydrogenase (Roche) and 0.5 μg/ml glycerol kinase (Roche) was added. Lipolysis was measured at optical density of 340 nm as the release of glycerol into the cell-free medium.

Heimann E., Nyman M, & Degerman E. (2014). Propionic acid and butyric acid inhibit lipolysis and de novo lipogenesis and increase insulin-stimulated glucose uptake in primary rat adipocytes. Adipocyte, 4(2), 81-88.

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Multi-color Flow Cytometric Analysis of Immune Cells

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Anti-CD3ε (145-2C11), anti-CD16/CD32 (2.4G2), PE-anti-CD73 (TY23), APC-anti-CD39 (TÜ66), FITC-anti-CD21/35 (7G6), PE- and APC-anti-PD-L2 (TY25), and FITC-anti-IgM^a (DS-1) were obtained from BD Biosciences (San Diego, CA, USA). Alexa Flour 647-anti-CD73 (TY11.8), FITC- and perCP-Cy5.5-anti-B220 (RA3-6B2), perCP-Cy5.5-F4/80, Alexa Fluor 647-anti-CD5 (53-7.3), APC-anti-CD93 (AA4.1), and APC-anti-Gr-1 (RB6-8C5) were obtained from Biolegend. PE-Cy7-anti-CD23 (2G8) was obtained from Abcam. PE-anti-IL-10 (JES5–16E3) was obtained from eBioscience (San Diego, CA). Anti-CD40 (1C10) was obtained from R&D Systems. Affinity-purified F(ab’)₂ fragments of goat anti-mouse IgM (anti-Ig) were obtained from Jackson Immunoresearch Laboratories. LPS from Salmonella typhimurium, and 5′-AMP, were obtained from Sigma Aldrich. Adenosine Deaminase (ADA) was obtained from Roche Diagnostics (Indianapolis, IN).

Kaku H., Cheng K.F., Al-Abed Y, & Rothstein T.L. (2014). A novel mechanism of B-cell mediated immune suppression through CD73-expression and adenosine production. Journal of immunology (Baltimore, Md. : 1950), 193(12), 5904-5913.

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Adenosine Receptor Ligands Characterization

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The ligands used were: adenosine and guanosine from Sigma-Aldrich (St. Louis, MO, USA); CGS21680 and SCH442416 (2-(2-furyl)-7-[3-(4-methoxyphenyl)propyl]-7H-pyrazolo [4,3-e]-[1,2,4]triazolo [1,5-c]pyrimidin-5-amine) from Tocris Bioscience (Ellisville, MI, USA). adenosine deaminase (ADA) was purchased from Roche Diagnostics (GmbH, Mannheim, Germany) and zardaverine from Calbiochem (San Diego, CA, USA). MRS7396, which is a selective fluorescent antagonist at the A_2AR derived from SCH442416, was previously described [19 (link)].

Lanznaster D., Massari C.M., Marková V., Šimková T., Duroux R., Jacobson K.A., Fernández-Dueñas V., Tasca C.I, & Ciruela F. (2019). Adenosine A1-A2A Receptor-Receptor Interaction: Contribution to Guanosine-Mediated Effects. Cells, 8(12), 1630.

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Measuring cAMP Levels in A2AR HEK-293T Cells

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cAMP accumulation was measured using the LANCE Ultra cAMP kit (PerkinElmer, Waltham, MA, USA) as previously described [27 ]. In brief, A_2AR HEK-293T cells were detached with accutase (Sigma-Aldrich) and incubated for 1 h at 22°C in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich) supplemented with 0.1% BSA, adenosine deaminase (ADA, 0.5 U/ml; Roche Diagnostics, GmbH, Mannheim, Germany) and Zardaverin (100 μM; Calbiochem, San Diego, CA, USA). Cells (1×10⁴ cells/200μl) were firstly incubated with vehicle or MRS7145 (10 μM or increasing concentrations) and then irradiated as above indicated. After 5 min, post-irradiated cells were incubated with CGS21680 (10 nM or increasing concentrations) during 30 min at 22°C. Eu-cAMP tracer and ULight^™-anti-cAMP reagents were prepared and added to the sample following manufacturer’s instructions. The 384-wells plate was incubated 1 h at 22°C in the dark and was then read on a POLARstar microplate reader (BMG Labtech, Durham, NC, USA). Measurements at 620 nm and 665 nm were used to detect the TR-FRET signal and the concomitant cAMP levels were calculated following manufacturer’s instructions. Data were fitted by non-linear regression using GraphPad Prism 6.01.

Taura J., Nolen E.G., Cabré G., Hernando J., Squarcialupi L., López-Cano M., Jacobson K.A., Fernández-Dueñas V, & Ciruela F. (2018). Remote control of movement disorders using a photoactive adenosine A2A receptor antagonist. Journal of controlled release : official journal of the Controlled Release Society, 283, 135-142.

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Glosensor™ cAMP Assay in HEK293G Cells

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Glosensor™ cAMP Human Embryonic Kidney 293 cell line (Hek293G) and GloSensor™ cAMP Reagent were obtained from Promega (Wisconsin, USA). Forskolin, NECA, Adenosine, XAC and SCH 58261 were from Sigma-Aldrich (Missouri, USA). PSB 603, ZM 241385, BAY 60-6583 and CGS 21680 hydrochloride were from Tocris Bioscience (Bristol, UK). Adenosine Deaminase (ADA) was from Roche (Mannheim, Germany). Hygromycin B was from Invitrogen (Paisley, UK). L-glutamine, trypsin and FCS were from Lonza (Verviers, Belgium). All other chemicals were from Sigma-Aldrich (Missouri, USA).

Goulding J., May L.T, & Hill S.J. (2018). Characterisation of endogenous A2A and A2B receptor-mediated cyclic AMP responses in HEK 293 cells using the GloSensor™ biosensor: Evidence for an allosteric mechanism of action for the A2B-selective antagonist PSB 603. Biochemical Pharmacology, 147, 55-66.

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Adenosine and PKG Signaling Modulation

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N6-cyclopentyladenosine (CPA) was purchased from Tocris, (9S,10R,12R)-2,3,9,10,11,12-Hexahydro-10-methoxy-2,9-dimet-hyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3′,2′,1′-kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acid methylester (KT5823) was from Santa Cruz Biotecnology, 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ) and NG-nitro-L-arginine methylester (L-NAME) were from Sigma and Adenosine deaminase (ADA) was from Roche. All other reagents used were from analytical grade.

Pinto I., Serpa A., Sebastião A.M, & Cascalheira J.F. (2016). The Role of cGMP on Adenosine A1 Receptor-mediated Inhibition of Synaptic Transmission at the Hippocampus. Frontiers in Pharmacology, 7, 103.

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Radioligand Binding Assay for hA2AAR

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The radioligand [³H] ZM241385 with a specific activity of 50 Ci × mmol⁻¹ was purchased from ARC Inc. (St. Louis, MO). Unlabelled ZM241385 was a gift from Dr. S.M. Poucher (Astra Zeneca, Macclesfield, UK). 5′-N-ethylcarboxamidoadenosine (NECA) was purchased from Sigma-Aldrich (Steinheim, Germany). LUF6632 was synthesized in our lab, as published previously [19 (link)]. Adenosine deaminase (ADA) was purchased from Boehringer Mannheim (Mannheim, Germany). Bicinchoninic acid (BCA) and BCA protein assay reagent were obtained from Pierce Chemical Company (Rockford, IL, USA). HEK293 cells stably expressing the hA_2A adenosine receptor (HEK293 hA_2AAR) were kindly provided by Dr. J Wang (Biogen/IDEC, Cambridge, MA, USA). All other chemicals were of analytical grade and obtained from standard commercial sources.

Yang X., Dong G., Michiels T.J., Lenselink E.B., Heitman L., Louvel J, & IJzerman A.P. (2016). A covalent antagonist for the human adenosine A2A receptor. Purinergic Signalling, 13(2), 191-201.

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Adenosine A1 Receptor Binding Assay

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[³H]-1,3-Dipropyl-8-cyclopentyl-xanthine ([³H]-DPCPX, specific activity 113.4 Ci · mmol⁻¹) was purchased from ARC, Inc. (St. Louis, MO). The Wheat Germ Agglutinin-Polyvinyl toluene (WGA-PVT) SPA beads (RPNQ0001) were purchased from PerkinElmer (Waltham, MA). Adenosine deaminase (ADA) was purchased from Boehringer Mannheim (Mannheim, Germany). 1,3-Dipropyl-8-cyclopentyl-xanthine (DPCPX, a selective hA₁R antagonist [23 (link)]), 8-cyclopentyl-3-N-[3-((3-(4-fluorosulphonyl)benzoyl)-oxy)-propyl]-1-N-propyl-xanthine (FSCPX, an irreversible hA₁R antagonist [24 (link)]), 2-chloro-N⁶-cyclopentyladenosine (CCPA, a selective hA₁R agonist [25 (link)]), N⁶-cyclopentyladenosine (CPA, a selective hA₁R agonist [25 (link)]), 5′-N-ethylcarboxamidoadenosine (NECA, a non-selective agonist for adenosine receptors [26 (link)]), and guanosine-5′-triphosphate (GTP) were purchased from Sigma (St. Louis, MO). BCA (bicinchoninic acid) protein assay kit was obtained from Pierce Chemical Company (Rockford, IL). LUF5834 (an hA₁R partial agonist) was synthesized in our laboratory as described previously [27 (link)]. Chinese hamster ovary (CHO) cells stably expressing the hA₁R were obtained from Prof. Steve Hill (University of Nottingham, UK). All other chemicals were of analytical grade and obtained from standard commercial sources.

Xia L., de Vries H., IJzerman A.P, & Heitman L.H. (2015). Scintillation proximity assay (SPA) as a new approach to determine a ligand’s kinetic profile. A case in point for the adenosine A1 receptor. Purinergic Signalling, 12(1), 115-126.

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