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Enzyme assays for synthesis and degradation of 2-5As and other 2'-5' oligonucleotides.

Poulsen JB, Kjær KH, Justesen J, Martensen PM - BMC Biochem. (2015)

Bottom Line: The 2-5As bind and activate the RNase L, an endoRNase degrading viral and cellular RNA leading to inhibition of viral replication.Whereas synthesis is mediated by the 2'-5' oligoadenylate synthetase family of enzymes, degradation seems to be orchestrated by multiple enzyme nucleases including phosphodiesterase 12, the ectonucleotide pyrophosphatase/phosphodiesterase 1 and the A-kinase anchoring protein 7.The nuclease reactions make use of the isolated 2'-5' oligoadenylates in producing a mixture of shorter reaction products, which are resolved by ion-exchange chromatography to determine the enzyme activities.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Genetics, Aarhus University, C.F. Møllers Allé 3, DK-8000, Aarhus C, Denmark. JESP@ssi.dk.

ABSTRACT

Background: The 5'-triphosphorylated, 2'-5'-linked oligoadenylate polyribonucleotides (2-5As) are central to the interferon-induced antiviral 2-5A system. The 2-5As bind and activate the RNase L, an endoRNase degrading viral and cellular RNA leading to inhibition of viral replication. The 2-5A system is tightly controlled by synthesis and degradation of 2-5As. Whereas synthesis is mediated by the 2'-5' oligoadenylate synthetase family of enzymes, degradation seems to be orchestrated by multiple enzyme nucleases including phosphodiesterase 12, the ectonucleotide pyrophosphatase/phosphodiesterase 1 and the A-kinase anchoring protein 7.

Results: Here we present assay tools for identification and characterization of the enzymes regulating cellular 2-5A levels. A procedure is described for the production of 2'-5' oligoadenylates, which are then used as substrates for development and demonstration of enzyme assays measuring synthetase and nuclease activities, respectively. The synthetase assays produce only a single reaction product allowing for very precise kinetic assessment of the enzymes. We present an assay using dATP and the A(pA)3 tetramer core as substrates, which requires prior isolation of A(pA)3. A synthetase assay using either of the dNTPs individually together with NAD(+) as substrates is also presented. The nuclease reactions make use of the isolated 2'-5' oligoadenylates in producing a mixture of shorter reaction products, which are resolved by ion-exchange chromatography to determine the enzyme activities. A purified human 2'-5' oligoadenylate synthetase and a purified human phosphodiesterase 12 along with crude extracts expressing those proteins, are used to demonstrate the assays.

Conclusions: This paper comprises an assay toolbox for identification and characterization of the synthetases and nucleases regulating cellular 2-5A levels. Assays are presented for both enzyme families. The assays can also be used to address a broader cellular role of the OAS enzymes, based on the multiple substrate specificity intrinsic to these proteins.

No MeSH data available.


Single-product OAS assay using dATP and A(pA)3 as substrates. Purified human recombinant His-OAS1 enzyme was incubated with dATP and A(pA)3 for 30 min at 37 °C, and the reactants and product resolved using a HiTrap Q column. In the figure, three separate chromatograms have been superimposed with the position of the respective molecules indicated. Blue curve: Profile from a reaction using His-OAS1 with dATP and A(pA)3 as substrates. Red and green curves: Control reactions using His-OAS1 together with dATP or A(pA)3 alone, respectively. Brown curve: Experimental salt gradient. Chromatograms were obtained with 254 nm as the absorbance wavelength. mAU, milli-absorbance unit and mS/cm, milliSiemens/centimeter
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Fig2: Single-product OAS assay using dATP and A(pA)3 as substrates. Purified human recombinant His-OAS1 enzyme was incubated with dATP and A(pA)3 for 30 min at 37 °C, and the reactants and product resolved using a HiTrap Q column. In the figure, three separate chromatograms have been superimposed with the position of the respective molecules indicated. Blue curve: Profile from a reaction using His-OAS1 with dATP and A(pA)3 as substrates. Red and green curves: Control reactions using His-OAS1 together with dATP or A(pA)3 alone, respectively. Brown curve: Experimental salt gradient. Chromatograms were obtained with 254 nm as the absorbance wavelength. mAU, milli-absorbance unit and mS/cm, milliSiemens/centimeter

Mentions: For demonstrating the assay we incubated the purified human His-OAS1 with the substrates dATP and A(pA)3 and allowed the reaction to proceed for 30 min. The reactants and products were resolved by anion-exchange chromatography using a HiTrap Q column and a gradient of increasing sodium chloride concentration. Compared with the controls dATP or A(pA)3 only, the product A(pA)3pdA exhibited a retention volume higher than A(pA)3 and lower than dATP, and formed a discrete peak (Fig. 2). We calculated the specific enzyme activity of the human OAS1 p42 for production of A(pA)3pdA to be 1.1 mmole/(sec*g).Fig. 2


Enzyme assays for synthesis and degradation of 2-5As and other 2'-5' oligonucleotides.

Poulsen JB, Kjær KH, Justesen J, Martensen PM - BMC Biochem. (2015)

Single-product OAS assay using dATP and A(pA)3 as substrates. Purified human recombinant His-OAS1 enzyme was incubated with dATP and A(pA)3 for 30 min at 37 °C, and the reactants and product resolved using a HiTrap Q column. In the figure, three separate chromatograms have been superimposed with the position of the respective molecules indicated. Blue curve: Profile from a reaction using His-OAS1 with dATP and A(pA)3 as substrates. Red and green curves: Control reactions using His-OAS1 together with dATP or A(pA)3 alone, respectively. Brown curve: Experimental salt gradient. Chromatograms were obtained with 254 nm as the absorbance wavelength. mAU, milli-absorbance unit and mS/cm, milliSiemens/centimeter
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4481073&req=5

Fig2: Single-product OAS assay using dATP and A(pA)3 as substrates. Purified human recombinant His-OAS1 enzyme was incubated with dATP and A(pA)3 for 30 min at 37 °C, and the reactants and product resolved using a HiTrap Q column. In the figure, three separate chromatograms have been superimposed with the position of the respective molecules indicated. Blue curve: Profile from a reaction using His-OAS1 with dATP and A(pA)3 as substrates. Red and green curves: Control reactions using His-OAS1 together with dATP or A(pA)3 alone, respectively. Brown curve: Experimental salt gradient. Chromatograms were obtained with 254 nm as the absorbance wavelength. mAU, milli-absorbance unit and mS/cm, milliSiemens/centimeter
Mentions: For demonstrating the assay we incubated the purified human His-OAS1 with the substrates dATP and A(pA)3 and allowed the reaction to proceed for 30 min. The reactants and products were resolved by anion-exchange chromatography using a HiTrap Q column and a gradient of increasing sodium chloride concentration. Compared with the controls dATP or A(pA)3 only, the product A(pA)3pdA exhibited a retention volume higher than A(pA)3 and lower than dATP, and formed a discrete peak (Fig. 2). We calculated the specific enzyme activity of the human OAS1 p42 for production of A(pA)3pdA to be 1.1 mmole/(sec*g).Fig. 2

Bottom Line: The 2-5As bind and activate the RNase L, an endoRNase degrading viral and cellular RNA leading to inhibition of viral replication.Whereas synthesis is mediated by the 2'-5' oligoadenylate synthetase family of enzymes, degradation seems to be orchestrated by multiple enzyme nucleases including phosphodiesterase 12, the ectonucleotide pyrophosphatase/phosphodiesterase 1 and the A-kinase anchoring protein 7.The nuclease reactions make use of the isolated 2'-5' oligoadenylates in producing a mixture of shorter reaction products, which are resolved by ion-exchange chromatography to determine the enzyme activities.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Genetics, Aarhus University, C.F. Møllers Allé 3, DK-8000, Aarhus C, Denmark. JESP@ssi.dk.

ABSTRACT

Background: The 5'-triphosphorylated, 2'-5'-linked oligoadenylate polyribonucleotides (2-5As) are central to the interferon-induced antiviral 2-5A system. The 2-5As bind and activate the RNase L, an endoRNase degrading viral and cellular RNA leading to inhibition of viral replication. The 2-5A system is tightly controlled by synthesis and degradation of 2-5As. Whereas synthesis is mediated by the 2'-5' oligoadenylate synthetase family of enzymes, degradation seems to be orchestrated by multiple enzyme nucleases including phosphodiesterase 12, the ectonucleotide pyrophosphatase/phosphodiesterase 1 and the A-kinase anchoring protein 7.

Results: Here we present assay tools for identification and characterization of the enzymes regulating cellular 2-5A levels. A procedure is described for the production of 2'-5' oligoadenylates, which are then used as substrates for development and demonstration of enzyme assays measuring synthetase and nuclease activities, respectively. The synthetase assays produce only a single reaction product allowing for very precise kinetic assessment of the enzymes. We present an assay using dATP and the A(pA)3 tetramer core as substrates, which requires prior isolation of A(pA)3. A synthetase assay using either of the dNTPs individually together with NAD(+) as substrates is also presented. The nuclease reactions make use of the isolated 2'-5' oligoadenylates in producing a mixture of shorter reaction products, which are resolved by ion-exchange chromatography to determine the enzyme activities. A purified human 2'-5' oligoadenylate synthetase and a purified human phosphodiesterase 12 along with crude extracts expressing those proteins, are used to demonstrate the assays.

Conclusions: This paper comprises an assay toolbox for identification and characterization of the synthetases and nucleases regulating cellular 2-5A levels. Assays are presented for both enzyme families. The assays can also be used to address a broader cellular role of the OAS enzymes, based on the multiple substrate specificity intrinsic to these proteins.

No MeSH data available.