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 language | English |
|---|---|
| Pages (from-to) | 602-610 |
| Number of pages | 9 |
| Journal | Analytical Biochemistry |
| Volume | 67 |
| Issue number | 2 |
| DOIs | |
| State | Published - Jan 1 1975 |
| Externally published | Yes |
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ASJC Scopus subject areas
- Biochemistry
- Biophysics
- Molecular Biology
Cite this
An assay for pyrimidine deoxyribonucleoside kinase using γ-32P-labeled ATP. / Dobersen, Michael J.; Greer, Sheldon.
In: Analytical Biochemistry, Vol. 67, No. 2, 01.01.1975, p. 602-610.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - An assay for pyrimidine deoxyribonucleoside kinase using γ-32P-labeled ATP
AU - Dobersen, Michael J.
AU - Greer, Sheldon
PY - 1975/1/1
Y1 - 1975/1/1
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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UR - http://www.scopus.com/inward/citedby.url?scp=0016537702&partnerID=8YFLogxK
U2 - 10.1016/0003-2697(75)90335-8
DO - 10.1016/0003-2697(75)90335-8
M3 - Article
VL - 67
SP - 602
EP - 610
JO - Analytical Biochemistry
JF - Analytical Biochemistry
SN - 0003-2697
IS - 2
ER -