TY - JOUR
T1 - An assay for pyrimidine deoxyribonucleoside kinase using γ-32P-labeled ATP
AU - Dobersen, Michael J.
AU - Greer, Sheldon
N1 - Funding Information:
’ This investigation was supported by Public Health Service Research Grant No. CA-12522 from the National Cancer Institute, Grant No. Al-12170 from the National Institute for Allergy and Infectious Diseases and Grant No. CA-14395 from the National Cancer Institute to the Comprehensive Cancer Center of Greater Miami. ’ The following abbreviations are used: DEAE, diethylaminoethyl cellulose; dT, thymidine; dC, deoxycytidine; BrdC. Sbromodeoxycytidine; PFU, plaque-forming units: PPO, 2.5-diphenyloxazole; POPOP, 1,4-bis-2(5phenyIoxazolyl)-benzene.
PY - 1975/8
Y1 - 1975/8
N2 - 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.
AB - 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.
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U2 - 10.1016/0003-2697(75)90335-8
DO - 10.1016/0003-2697(75)90335-8
M3 - Article
C2 - 169711
AN - SCOPUS:0016537702
VL - 67
SP - 602
EP - 610
JO - Analytical Biochemistry
JF - Analytical Biochemistry
SN - 0003-2697
IS - 2
ER -