An isotopic assay of dUTPase activity based on coupling with thymidylate synthase.
A new rapid, sensitive and convenient procedure is presented allowing determination of dUTPase activity. With [5-(3)H]dUTP used as the substrate, dUTPase, converts it to the corresponding monophosphate and is coupled with thymidylate synthase-catalyzed reaction, resulting in tritium release from [5-(3)H]dUMP. Following charcoal absorption of the labeled nuleotides, radioactivity of tritiated water is determined. The new assay was tested to show comparable results with a previously described assay, based on measuring dUTPase-catalyzed [5-(3)H]dUMP production.
Gołos
B
B
Nencki Institute of Experimental Biology, Polish Academy of Sciences, Warszawa.
Rode
W
W
D000818:Animals; D003856:Deoxyuracil Nucleotides / Q000378:metabolism; D066298:In Vitro Techniques; D008099:Liver / Q000201:enzymology; D008115:Liver Regeneration; D011755:Pyrophosphatases / Q000032:analysis* / Q000378:metabolism*; D051381:Rats; D012680:Sensitivity and Specificity; D013379:Substrate Specificity; D013940:Thymidylate Synthase / Q000378:metabolism*; D014316:Tritium
D003856:Deoxyuracil Nucleotides; D014316:Tritium; C027078:deoxyuridine triphosphate; D013940:Thymidylate Synthase; D011755:Pyrophosphatases; C020020:dUTP pyrophosphatase