| Autor: |
Bierau, Jörgen, Leen, René, van Gennip, Albert H., Caron, Huib N., van Kuilenburg, André B. P. |
| Přispěvatelé: |
Amsterdam Gastroenterology Endocrinology Metabolism, Cancer Center Amsterdam, Laboratory Genetic Metabolic Diseases, Amsterdam Public Health, Paediatric Oncology |
| Jazyk: |
angličtina |
| Rok vydání: |
2004 |
| Zdroj: |
Journal of chromatography. B, Analytical technologies in the biomedical and life sciences, 805(2), 339-346. Elsevier |
| ISSN: |
1570-0232 |
| Popis: |
A non-radioactive procedure to measure the deoxycytidine kinase (dCK) activity in crude cell free homogenates was developed. 2-Chlorodeoxyadenosine (CdA) was used as the substrate for dCK and was separated from its product 2-chlorodeoxyadenosine-5'-monophosphate (CdAMP) by reversed-phase HPLC. A complete separation of CdA and its metabolites was achieved in 30 min. The minimum amount of CdAMP that could be detected was 1 pmol. The assay was linear with reaction times up to at least 3h. With respect to the protein concentration, the reaction was linear with protein concentrations up to 760 microg/ml in the assay. An amount of 8 x 10(3) cells was already sufficient to determine the specific dCK activity in SK-N-BE(2)c cells. CdA was not only converted to CdAMP but also to 2-chloroadenine and, surprisingly, also to 2-chlorodeoxyinosine, in MOLT-3 cells. The deamination of CdA was completely inhibited by deoxycoformycin, which clearly demonstrates that CdA is a substrate for adenosine deaminase |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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