An assay for pyrimidine deoxyribonucleoside kinase using γ-32P-labeled ATP

Michael J. Dobersen, Sheldon Greer

Research output: Contribution to journalArticle

18 Scopus citations

Abstract

A rapid, simple and sensitive assay for pyrimidine deoxyribonucleoside kinase is described. After phosphorylation of unlabeled nucleoside substrate through the transfer of the γ-phosphate of [γ-32P]ATP, the reaction mixture is subjected to 1 n HCl at 100°C. The β- and γ-phosphates of unreacted ATP are hydrolyzed to inorganic phosphate while the 5′-phosphate of the pyrimidine deoxyribonucleotide product is not hydrolyzed. Radioactive phosphate remaining in the supernatant fluid after precipitation of inorganic phosphate corresponds to product and is measured by liquid scintillation spectrometry. Evidence is presented suggesting that pyrimidine and purine ribonucleoside kinase activity can also be determined by this assay.

Original languageEnglish (US)
Pages (from-to)602-610
Number of pages9
JournalAnalytical Biochemistry
Volume67
Issue number2
DOIs
StatePublished - Aug 1975

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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