A method for quantification of nucleotides and nucleotide analogues in thymidine kinase assays using lanthanum phosphate coprecipitation.
Current methodologies for quantifying radiolabeled nucleoside monophosphates and nucleoside analogues result in high retention of unphosphorylated guanosine nucleosides in the case of lanthanum chloride precipitation or inconsistent retention of nucleotides in the case of DEAE cellulose filter papers. This study describes an innovative method for quantifying thymidine kinase (TK) activity that is compatible with both purine and pyrimidine nucleoside analogues by using lanthanum phosphate coprecipitation at pH 4.0. This methodology maintains quantitative precipitation of nucleoside monophosphates and yields minimal background binding from a variety of nucleoside analogues. In addition, use of PCR thermocyclers enhances the temporal precision of TK assays. This method was shown to be useful for assaying TK activity in a broad range of biochemically relevant systems, including purified enzymes, stable cell lines, and virally infected cells. Use of this methodology should aid researchers in the evaluation of novel nucleoside analogues and TK enzymes while decreasing radioactive waste, minimizing assay time, increasing accuracy, and enhancing dynamic range.
Gammon
S T
ST
Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, MO 63110, USA.
Bernstein
M
M
Schuster
D P
DP
Piwnica-Worms
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D
D002478:Cells, Cultured; D011232:Chemical Precipitation; D018259:Herpesvirus 1, Human / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D007811:Lanthanum / Q000378:metabolism; D009711:Nucleotides / Q000032:analysis*; D010710:Phosphates / Q000737:chemistry*; D016133:Polymerase Chain Reaction; D012015:Reference Standards; D015203:Reproducibility of Results; D013937:Thymidine Kinase / Q000302:isolation & purification / Q000378:metabolism*; D013997:Time Factors
D009711:Nucleotides; D010710:Phosphates; C028521:lanthanum chloride; C476779:lanthanum(III) phosphate; D007811:Lanthanum; D013937:Thymidine Kinase