Phosphodiesterase 1

Manufactured by Worthington

Sourced in United States

Phosphodiesterase I is an enzyme that catalyzes the hydrolysis of phosphodiester bonds in nucleic acids, such as DNA and RNA. It is commonly used in molecular biology and biochemistry applications for the analysis and manipulation of nucleic acids.

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Lab products found in correlation

Nuclease p1, by Fujifilm (6 mentions) Nuclease p1, by Merck Group (6 mentions) Alkaline phosphatase, by Merck Group (5 mentions) Sep pak c18 cartridge, by Waters Corporation (5 mentions) Bovine spleen phosphodiesterase 2, by Merck Group (3 mentions) Dnase 1, by Merck Group (3 mentions) Nicotinamide, by Merck Group (3 mentions) Ribonuclease a, by Merck Group (3 mentions) Adp ribosyl cyclase, by Merck Group (3 mentions) Ammonium formate, by Merck Group (3 mentions) Protease, by Qiagen (3 mentions) Lc ms grade formic acid, by Thermo Fisher Scientific (3 mentions) Bacterial alkaline phosphatase type 3 escherichia coli, by Merck Group (2 mentions) Hplc grade acetonitrile, by Fujifilm (2 mentions) Phosphodiesterase 2, by Worthington (2 mentions) Ammonium bicarbonate, by Merck Group (2 mentions) Alcohol dehydrogenase, by Merck Group (2 mentions) Diaphorase, by Merck Group (2 mentions) Bicine, by Merck Group (2 mentions) Resazurin, by Merck Group (2 mentions)

24 protocols using phosphodiesterase 1

Reagents for DNA and RNA Analysis

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o-Toluidine hydrochloride (T536215; purity, 97%) was obtained from Toronto Research Chemicals, Toronto, ON, Canada, p-toluidine (PT) hydrochloride (T0303; purity, >98%) was obtained from Tokyo Chemical Industry, Tokyo, Japan, and aniline (ANL) hydrochloride (017-04092; purity, 97%) was obtained from Wako Pure Chemical Industries, Osaka, Japan. AAOT (A1020; purity, >98%) and apocynin (H0261; purity, >98%) were obtained from the Tokyo Chemical Industry. Nuclease P1 and HPLC-grade acetonitrile were purchased from Wako Pure Chemical Industries, Ltd. Phosphodiesterase I was purchased from Worthington Biochem (Lakewood, NJ, USA). Bovine spleen Phosphodiesterase II, DNase I, and bacterial alkaline phosphatase Type III (Escherichia coli) were purchased from SigmaAldrich (St. Louis, MO, USA). All other chemicals used were of analytical grade and purchased from Wako Pure Chemical Industries, Ltd.

Suzuki S., Gi M., Komiya M., Obikane A., Vachiraarunwong A., Fujioka M., Kakehashi A., Totsuka Y, & Wanibuchi H. (2023). Evaluation of the Mechanisms Involved in the Development of Bladder Toxicity following Exposure to Occupational Bladder Cancer Causative Chemicals Using DNA Adductome Analysis. Biomolecules, 14(1), 36.

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Purification and Characterization of Biomolecules

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All chemicals and reagents used were obtained at the highest purity available and were used without further purification unless stated. Benzonase, bacterial alkaline phosphatase, butylated hydroxytoluene, acetonitrile, and buffer salts were purchased from Sigma-Aldrich. Coformycin was obtained from the National Cancer Institute. Phosphodiesterase I was purchased from Worthington. Tetrahydrouridine was purchased from Calbiochem. Water purified through a Milli-Q system (Millipore) was used throughout our studies. Sartorius Vivaspin 500-brand centrifugal filter units were used for dialysis and sample concentration.

Lin C.J., Smibert P., Zhao X., Hu J.F., Ramroop J., Kellner S.M., Benton M.A., Govind S., Dedon P.C., Sternglanz R, & Lai E.C. (2015). An extensive allelic series of Drosophila kae1 mutants reveals diverse and tissue-specific requirements for t6A biogenesis. RNA, 21(12), 2103-2118.

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DNA Adduct Analysis Procedure

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PhIP-HCl was purchased from the Nard Institute (Osaka, Japan), and its purity was confirmed to be >99% by HPLC. AA was obtained from Sigma Aldrich (St. Louis, MO, United States). Nuclease P1 was purchased from Wako Pure Chemical Industries (Osaka, Japan). Phosphodiesterase I was purchased from Worthington. Nicotinamide, bovine spleen Phosphodiesterase II, DNase I, and bacterial alkaline phosphatase type III (E. coli) were purchased from Sigma Aldrich (St. Louis, MO, United States). Difco™ Nutrient Broth and Cofactor-I were acquired from Becton, Dickinson and Company (Sparks, MD, United States), and Oriental Yeast Co., Ltd (Tokyo, Japan), respectively. The S9 mix was purchased from the IEDA Trading Corporation (Tokyo, Japan). All other chemicals used in the study were of analytical grade and were purchased from Wako Pure Chemical Industries.

Komiya M., Ishigamori R., Naruse M., Ochiai M., Miyoshi N., Imai T, & Totsuka Y. (2021). Establishment of Novel Genotoxicity Assay System Using Murine Normal Epithelial Tissue-Derived Organoids. Frontiers in Genetics, 12, 768781.

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Oligodeoxynucleotide Synthesis and Enzymatic Assays

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All nucleoside phosphoramidites including 5-methyl-dC, 5-hydroxymethyl-dC, 5-formyl-dC-III, 5-carboxy-dC, Ac-dC, dT, dA, dG, and dmf-dG, reagents, and controlled pore glass solid support for oligodeoxynucleotide synthesis were acquired from Glen Research Corporation (Sterling, VA). Human recombinant DNA methyltransferase 1 (DNMT1) and Δ580-DNMT1^{8 (link)} (missing the PCNA,^{23 (link)} DNMT3A/B interaction domains^{23 (link), 24 (link)}) were purchased from New England BioLabs (Ipswich, MA). Phosphodiesterase I, Phosphodiesterase II, and DNase I were acquired from Worthington Biochemical Corporation (Lakewood, NJ). Bovine intestinal alkaline phosphatase was procured from Sigma Aldrich Chemical Company (Milwaukee, WI). All remaining laboratory chemicals and solvents were purchased from ThermoFisher Scientific (Waltham, MA) and Sigma-Aldrich (Milwaukee, WI). The synthesis of ¹³C₁₀¹⁵N₂-5-Methyl-2′-deoxycytidine was described previously.^{22 (link)}

Seiler C.L., Fernandez J., Koerperich Z., Andersen M.P., Kotandeniya D., Nguyen M.E., Sham Y.Y, & Tretyakova N.Y. (2018). Maintenance DNA methyltransferase activity in the presence of oxidized forms of 5-methylcytosine: structural basis for ten eleven translocation-mediated DNA demethylation. Biochemistry, 57(42), 6061-6069.

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Nuclease P1 and Phosphodiesterase Analysis

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1,4-Dioxane was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan; purity >99.9%). Nuclease P1 and HPLC-grade acetonitrile were also purchased from Wako Pure Chemical Industries, Ltd. Phosphodiesterase I was purchased from Worthington Biochem (Lakewood, NJ, U.S.A.). Bovine spleen Phosphodiesterase II, DNase I, and bacterial alkaline phosphatase Type III (Escherichia coli) were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). All other chemicals used were of analytical grade and purchased from Wako.

TOTSUKA Y., MAESAKO Y., ONO H., NAGAI M., KATO M., GI M., WANIBUCHI H., FUKUSHIMA S., SHIIZAKI K, & NAKAGAMA H. (2020). Comprehensive analysis of DNA adducts (DNA adductome analysis) in the liver of rats treated with 1,4-dioxane. Proceedings of the Japan Academy. Series B, Physical and Biological Sciences, 96(5), 180-187.

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Comprehensive Analytical Protocol Catalog

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Ammonium bicarbonate, ammonium acetate, diepoxybutane, bis-(2-chloroethyl)amine hydrochloride (nornitrogen mustard), mechlorethamine, d,l -1,2,3,4-diepoxybutane, phenylmethanesulfonyl fluoride (PMSF), Boc-L-cysteine (Boc-Cys-OH), triflouroacetic acid (TFA), leupeptin, pepstatin, aprotinin, methoxyamine, UCN-01, dithiothreitol (DTT), iodoacetamide, chloroform, ribonuclease A, deoxyribonuclease I, and alkaline phosphatase were purchased from Sigma (St. Louis, MO). Phosphodiesterase I and Phosphodiesterase II were obtained from Worthington Biochemical Corporation (Lakewood, NJ). UltraPure buffer-saturated phenol was obtained from Invitrogen (Carlsbad,CA). Mass spectrometry grade trypsin was purchased from Promega (Madison, WI). Proteinase K was obtained from New England Biolabs (Beverly, MA). Cell Lysis Solution and Protein Precipitation solution were purchased from Qiagen (Valencia, Ca). Phosphoramide mustard was obtained from iTT GmbH/Niomech (Bielefeld, Germany).

Groehler AS I.V., Villalta P.W., Campbell C, & Tretyakova N. (2016). Covalent DNA-Protein Cross-linking by Phosphoramide Mustard and Nornitrogen Mustard in Human Cells. Chemical research in toxicology, 29(2), 190-202.

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Liquid Chromatography-Mass Spectrometry Protocol for tRNA Analysis

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The isolated tRNAs were digested with RNase T₁ (Ambion) and analyzed by capillary liquid chromatography (LC)/nano-electron spray ionization mass spectrometry (ESI-MS), as described (9 (link),42 (link),43 (link)). SI-labeled nucleoside analysis by LC/MS was performed as described (44 (link),45 (link)). For nucleoside analysis of HeLa cells cultured under taurine-depleted condition, 5 μg of tRNA fraction was digested as follows: 10 μl reaction mixture consisting of 20 mM ammonium acetate (pH 5.3) and 0.01 units/μl nuclease P1 (Wako Pure Chemical Industries) was incubated at 37°C for 30 min, followed by adding 0.5 μl of 1 M ammonium bicarbonate (pH 8.0) and 0.25 units of phosphodiesterase I (Worthington Biochemical Corporation) and incubated at 37°C for 30 min, then 0.1 units of alkaline Phosphatase (Escherichia coli C75) (Takara Bio) was added and further incubated at 37°C for 30 min. The digests were subjected to hydrophilic interaction liquid chromatography (HILIC)/MS analysis as described (46 (link)). For detection of THF derivatives bound to Thermotoga maritima MnmE, 175 μg of recombinant protein was directly injected into the LC/MS under the same conditions used for the nucleoside analysis.

Asano K., Suzuki T., Saito A., Wei F.Y., Ikeuchi Y., Numata T., Tanaka R., Yamane Y., Yamamoto T., Goto T., Kishita Y., Murayama K., Ohtake A., Okazaki Y., Tomizawa K., Sakaguchi Y, & Suzuki T. (2018). Metabolic and chemical regulation of tRNA modification associated with taurine deficiency and human disease. Nucleic Acids Research, 46(4), 1565-1583.

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Quantification of Total Nucleosides with ct6A

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Total nucleosides containing ct⁶A were usually prepared by neutral one-step digestion of total RNA (31 (link)). Total RNA (40 μg) was digested at 37°C for 1.5 h in 20 mM HEPES-KOH (pH 7.1) containing 0.1 U Nuclease P1 (Wako Pure Chemical Industries, Ltd.) and 0.08 U bacterial alkaline phosphatase (BAP) (E. coli C75, Wako Pure Chemical Industries, Ltd.). For the analysis of the trmO mutation, total RNA was completely digested by three-step digestion, as follows. Total RNA (40 μg) was incubated at 37°C for 1 h in 25 mM NH₄OAc (pH 5.3) containing 0.1 unit Nuclease P1. Thereafter, 0.1 volume of 1 M ammonium bicarbonate (pH 8.0) with 0.127 units of phosphodiesterase I (PDase I) (Worthington Biochemical Corporation) was added to the mixture, followed by incubation at 37°C for 1 h. Finally, 0.08 U BAP was added, and the sample was incubated at 37°C for 1.5 h. Prior to use, Nuclease P1, BAP and phosphodiesterase I were dialyzed against deionized water and stored at -30°C.

Kimura S., Miyauchi K., Ikeuchi Y., Thiaville P.C., de Crécy-Lagard V, & Suzuki T. (2014). Discovery of the β-barrel–type RNA methyltransferase responsible for N6-methylation of N6-threonylcarbamoyladenosine in tRNAs. Nucleic Acids Research, 42(14), 9350-9365.

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Phosphodiesterase and Alkaline Phosphatase Assay

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MC was generously gifted by Professor Maria Tomasz. Phosphodiesterase I (snake venom diesterase (SVD), Crotalus adamanteus venom, E.C. 3.1.4.1.) and alkaline phosphatase (Escherichia coli, EC 3.1.3.1) were obtained from Worthington Biochemical Corp (Freehold, NJ). Nuclease P₁ (penicillium citrinum, EC 3.1.30.1) was from Sigma Life Sciences (St. Louis, MO). Sep-Pak C-18 cartridges were purchased from Waters Corp (Milford, MA). Oligonucleotides were purchased from Midland Certified Reagent (Midland, TX).

Zacarias O., Petrovic A.G., Abzalimov R., Pradhan P, & Champeil E. (2021). Synthesis of Oligonucleotides Containing Trans Mitomycin C DNA Adducts at N6 of Adenine and N2 of Guanine. Chemistry (Weinheim an der Bergstrasse, Germany), 27(57), 14263-14272.

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Purification of Metabolic Enzymes

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Hexokinase, glucose-6-phosphate dehydrogenase, glutamate dehydrogenase, 6-phosphogluconate dehydrogenase, phosphoriboisomerase, myokinase, pyruvate kinase and ADP-ribosylcyclase were purchased from Sigma. Phosphodiesterase I was purchased from Worthington Biochemical. Alkaline phosphatase was purchased from New England Biolabs. Human PNP and NRK1 were purchased from Novus Biologicals. E. coli phosphoribosylpyrophosphate synthetase was expressed and purified using a published method.³² Nicotinate phosphoribosyltransferase was purified as described before.^{33 (link)}

Tran A., Yokose R, & Cen Y. (2018). Chemo-enzymatic Synthesis of Isotopically Labeled Nicotinamide Riboside. Organic & biomolecular chemistry, 16(19), 3662-3671.

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