Bromovinyl-deoxyuridine: A selective substrate for mitochondrial thymidine kinase in cell extracts
Journal Article
·
· Biochemical and Biophysical Research Communications
- Department of Biology, University of Padova, 35131 Padova (Italy)
- Instituto de Quimica Medica (CSIC), 28006 Madrid (Spain)
- Rega Institute for Medical Research, K. U. Leuven, B-3000 Leuven (Belgium)
Cellular models of mitochondrial thymidine kinase (TK2) deficiency require a reliable method to measure TK2 activity in whole cell extracts containing two interfering deoxyribonucleoside kinases, thymidine kinase 1 (TK1) and deoxycytidine kinase. We tested the value of the thymidine analog (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) as a TK2-specific substrate. With extracts of OSTTK1{sup -} cells containing TK2 as the only thymidine kinase and a highly specific TK2 inhibitor we established conditions to detect the low TK2 activity commonly present in cells. With extracts of TK1-proficient osteosarcoma cells and normal human fibroblasts we showed that BVDU, but not 1-({beta}-D-arabinofuranosyl)thymine (Ara-T), discriminates TK2 activity even in the presence of 100-fold excess TK1. A comparison with current procedures based on TK2 inhibition demonstrated the better performance of the new TK2 assay. When cultured human fibroblasts passed from proliferation to quiescence TK2 activity increased by 3-fold, stressing the importance of TK2 function in the absence of TK1.
- OSTI ID:
- 20798959
- Journal Information:
- Biochemical and Biophysical Research Communications, Journal Name: Biochemical and Biophysical Research Communications Journal Issue: 1 Vol. 344; ISSN 0006-291X; ISSN BBRCA9
- Country of Publication:
- United States
- Language:
- English
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