Improved measurement of thymidylate synthetase activity by a modified tritium-release assay

R. Douglas Armstrong, Robert B. Diasio

Research output: Contribution to journalArticle

20 Scopus citations

Abstract

Accurate quantitation of thymidylate synthetase activity using a tritium-release assay is dependent upon measurement of only that tritium released from deoxy[5-3H]uridine monophosphate ([3H]dUMP) during the biosynthesis of thymidylate. Removal of remaining [3H]dUMP on completion of the assay by charcoal adsorption and correction for the nonenzymatic release of tritium are necessary. Although over 99% of [3H]dUMP is removed immediately following addition of charcoal, these studies demonstrate that sufficient [3H]dUMP can remain to prevent accurate measurement of low levels of thymidylate synthetase activity. By delaying measurement of radioactivity for at least 24 h following addition of charcoal, this problem is minimized. To account for nonenzymatic release of tritium, a blank containing enzyme extract with omission of ±,l-5,10-methylenetetrahydrofolate is demonstrated to be more effective than the commonly used blank in which water is substituted for enzyme extract. In samples containing 5-fluoro-2′-deoxyuridine monophosphate (FdUMP), a potent inhibitor of thymidylate synthetase activity, an alternative blank containing a high concentration of FdUMP (approximately 1mM) is useful in demonstrating a theoretical maximal or complete inhibition of thymidylate synthetase activity.

Original languageEnglish (US)
Pages (from-to)141-147
Number of pages7
JournalJournal of Biochemical and Biophysical Methods
Volume6
Issue number2
DOIs
StatePublished - Jun 1982

Keywords

  • 5-fluorouracil
  • thymidylate synthetase
  • tritium-release assay

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry

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