A simple and sensitive ribonucleotide reductase assay

Ambrose Y. Jong, Kefei Yu, Bingsen Zhou, Tomǎs Frgala, Patrick Reynolds, Yun Yen

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17 引文 斯高帕斯(Scopus)

摘要

Ribonucleotide reductase (RR) is a key regulatory enzyme in the DNA synthesis pathway and is the target of the cancer chemotherapeutic agent hydroxyurea. The study of RR is significantly hindered by the tedious and labor-intensive nature of enzymatic assay. In this report, we present a novel RR assay in which detection of the deoxyribonucleotides produced by RR occurs via coupling to the DNA polymerase reaction, and is enhanced by using RNase to degrade endogenous RNA. Cell extracts from various cell lines were treated with RNase and then reacted with ATP and radioactive ribonucleotide diphosphate as the substrate. Incorporation of the radioactive substrate [14C]CDP into DNA was linear over 30 min and was linear with the amount of extract, which provided RR activity. The reaction was inhibited by hydroxyurea and required Mg2+ and ATP, suggesting that the assay is specific to RR activity. While RR activities determined by our method and by a conventional method were comparable, this novel method proved to be simpler faster, more sensitive and less expensive. In addition, assay of the RR activity for multiple samples can easily be performed simultaneously. It is superior to other RR assays in all aspects.

原文英語
頁(從 - 到)62-68
頁數7
期刊Journal of Biomedical Science
5
發行號1
DOIs
出版狀態已發佈 - 一月 1 1998
對外發佈Yes

ASJC Scopus subject areas

  • Endocrinology, Diabetes and Metabolism
  • Molecular Biology
  • Clinical Biochemistry
  • Cell Biology
  • Biochemistry, medical
  • Pharmacology (medical)

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