Purine nucleoside phosphorylase

Manufactured by Merck Group

Sourced in United States

Purine nucleoside phosphorylase is an enzyme that catalyzes the reversible phosphorolytic cleavage of purine nucleosides, such as inosine and guanosine, to their respective purine bases and ribose-1-phosphate. It is an important component in the purine salvage pathway and plays a role in the regulation of purine levels in cells.

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Lab products found in correlation

Inorganic pyrophosphatase, by Merck Group (2 mentions) Xanthine oxidase, by Merck Group (2 mentions) Sypro orange, by Merck Group (1 mentions) Dna polymerase, by New England Biolabs (1 mentions) Isopropyl β d 1 thiogalactopyranoside (iptg), by Thermo Fisher Scientific (1 mentions) Nickel nitrilotriacetic acid ni nta, by Qiagen (1 mentions) Cdpcho, by Merck Group (1 mentions) Dna purification kit, by Merck Group (1 mentions) Restriction enzyme, by New England Biolabs (1 mentions) Protease inhibitor cocktail tablet, by Roche (1 mentions) Icon nmr, by Bruker (1 mentions) Topspin 3, by Bruker (1 mentions) Lambda 45 spectrophotometer, by PerkinElmer (1 mentions) Origin 2019, by OriginLab (1 mentions) Origin 2021, by OriginLab (1 mentions) Jm109, by Takara Bio (1 mentions) Horseradish peroxidase, by Merck Group (1 mentions)

6 protocols using purine nucleoside phosphorylase

Purification and Characterization of Enzymes

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Restriction enzymes and DNA polymerases were obtained from New England Biolabs (Ipswich, MA, USA). Isopropyl β-D-1-thiogalactopyranoside (IPTG) was obtained from Fisher Scientific GmbH (Schwerte, Germany). Nickel-nitrilotriacetic acid (Ni-NTA) was from Qiagen (Düsseldorf, Germany), protease inhibitor cocktail tablets were purchased from Roche (Basel, Switzerland). CTP, CDPCho, Sypro Orange, inorganic pyrophosphatase, purine nucleoside phosphorylase, DNA purification kit and antibiotics were purchased from Sigma-Aldrich (St Louis, MO, USA). Phosphocholine chloride sodium salt hydrate (further termed as ChoP) was from TCI Europe N.V. (Antwerp, Belgium). MESG (7-methyl-6-thioguanosine) was obtained from Berry and Associates (Dexter, MI, USA). All other chemicals were of analytical grade of the highest purity available.

Marton L., Nagy G.N., Ozohanics O., Lábas A., Krámos B., Oláh J., Vékey K, & Vértessy B.G. (2015). Molecular Mechanism for the Thermo-Sensitive Phenotype of CHO-MT58 Cell Line Harbouring a Mutant CTP:Phosphocholine Cytidylyltransferase. PLoS ONE, 10(6), e0129632.

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Phosphorolysis of NR+ Cl- Compounds

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Phosphorolysis of NR⁺ Cl⁻ and its glycine conjugate (12a, Gly-NR) by PNP (Purine Nucleoside Phosphorylase, Sigma Aldrich) was performed in HEPES buffer, containing KH₂PO₄ and 10% D₂O at 25 °C and was monitored by ¹H NMR. All spectra were obtained at 300 K on a Bruker AscendTM 400 MHz ultra-shielded spectrometer (Bruker Biospin) operating at 400.13 MHz for protons. TopSpin 3.2 (Bruker BioSpin) was used for all NMR spectral acquisition and pre-processing. The automation of sample submission was performed using ICON-NMR (Bruker BioSpin). Incubations were conducted in the NMR tube which contained a final volume of 505 μl : 450 μl HEPES buffer (100.0 mM, pH 7.0), containing 100 mM KH₂PO₄, 50.0 μl NR-Cl or NR glycine conjugate (50.0 mM in 1 mL D₂O) and when appropriate 5 μl PNP (1 mg dissolved in 50 μl HEPES buffer) and measurements taken at t = 0, 20 min and 6 h (ns = 128). For each independent experiment, freshly prepared solutions of NR-Cl and NR glycine conjugate in HEPES buffer, containing 100 mM KH₂PO₄ and 50 μl D₂O, were used.

Hayat F, & Migaud M.E. (2020). Nicotinamide riboside–amino acid conjugates that are stable to purine nucleoside phosphorylase. Organic & biomolecular chemistry, 18(15), 2877-2885.

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Dephosphorylation of SAD-A by PP2Cα

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Dephosphorylation of the LKB1-activated SAD-A proteins by PP2Cα was measured using a continuous spectrophotometric assay62 (link). The assay was performed at 25 °C in a coupled enzyme system containing 50 mM MOPS, pH 7.0, 100 mM NaCl, 0.1 mM EDTA, 10 mM MgCl₂, 100 μM 7-methyl-6-thioguanosine (MESG, Berry & Associates), and 0.1 mg ml⁻¹ purine nucleoside phosphorylase (Sigma). This coupled system uses purine nucleoside phosphorylase and its chromogenic substrate MESG to monitor the production of inorganic phosphate. The reactions were initiated by adding PP2Cα, and the continuous absorbance changes at 360 nm were recorded with a PerkinElmer LAMBDA 45 spectrophotometer equipped with a magnetic stirrer in the cuvette holder. The concentration of MESG was determined at 331 nm using a molar extinction coefficient of 32,000 M⁻¹ cm⁻¹, and quantification of phosphate release was measured at 360 nm using the extinction coefficient of 11,200 M⁻¹ cm⁻¹ (ref. 63 (link)).

Wu J.X., Cheng Y.S., Wang J., Chen L., Ding M, & Wu J.W. (2015). Structural insight into the mechanism of synergistic autoinhibition of SAD kinases. Nature Communications, 6, 8953.

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Continuous Photometric Assay for Enzyme Kinetics

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Enzyme activities were measured under steady‐state conditions using a coupled photometric assay, which allowed for the continuous measurement of the released phosphate (Suárez et al., 2012 (link)). The reaction mixture contained 100 mM Tris/HCl (pH 7.8), 5 mM MgCl₂, 0.5 mM inosine, 0.25 U/mL purine nucleoside phosphorylase (Sigma‐Aldrich), 2.5 U/mL xanthine oxidase (Sigma‐Aldrich), and variable concentrations of the tested phosphorylated substrates. The reactions were started by addition of the respective enzyme. Kinetic parameters were calculated from the initial ascending slopes by curve fitting with the Michaelis–Menten equation (Origin 2019 and Origin 2021, OriginLab).

Kinateder T., Mayer C., Nazet J, & Sterner R. (2024). Improving enzyme functional annotation by integrating in vitro and in silico approaches: The example of histidinol phosphate phosphatases. Protein Science : A Publication of the Protein Society, 33(2), e4899.

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Enzymatic Inosine Hydrolysis Assay

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Purine nucleoside phosphorylase (human, recombinantly expressed in E. coli) was purchased from Sigma-Aldrich. The reaction solution for inosine hydrolysis consisted of 50 mM KPB (pH 7.4), with varying concentrations of inosine and oxypurinol. The reaction was quenched with perchloric acid and neutralized by K₂CO₃, with hypoxanthine as products analyzed by HPLC.

Sekine M., Okamoto K., Pai E.F., Nagata K., Ichida K., Hille R, & Nishino T. (2023). Allopurinol and oxypurinol differ in their strength and mechanisms of inhibition of xanthine oxidoreductase. The Journal of Biological Chemistry, 299(9), 105189.

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E. coli and Yeast Strain Cultivation

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The E. coli strains used in this study were JM109 (Takara Bio Inc., Shiga, Japan) and BL21(DE3) (Novagen, Abingdon, UK), which were grown in Luria-Bertani (LB) medium (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) with aeration at 37 °C. Saccharomyces cerevisiae OP-3C was as described previously. 21 Plasmid pET-42b was from Novagen. Inorganic pyrophosphatase, purine nucleoside phosphorylase, xanthine oxidase and horseradish peroxidase were purchased from Sigma-Aldrich. Potato acid phosphatase was purchased from GE Healthcare UK Ltd. (Buckinghamshire, UK). Restriction enzymes and other DNA-modifying enzymes were purchased from New England Biolabs (Beverly, MA, USA) or Takara Bio Inc. and used as described by the manufacturers.

Inokoshi J., Nakamura Y., Komada S., Komatsu K., Umeyama H, & Tomoda H. (2016). Inhibition of bacterial undecaprenyl pyrophosphate synthase by small fungal molecules. The Journal of antibiotics, 69(11).

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