Bromovinyl-deoxyuridine: A selective substrate for mitochondrial thymidine kinase in cell extracts
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- 作者：Elisa Franzolin ; Chiara Rampazzo ; Marí ; a-Jesú ; s Pé ; rez-Pé ; rez ; Ana-Isabel Herná ; ndez ; Jan Balzarini and Vera Bianchi
- 关键词：Thymidine kinase 2 ; Thymidine analogs ; Mitochondrial DNA precursors ; TK2-deficiency ; Mitochondrial diseases ; Mitochondrial DNA depletion syndromes ; Enzyme assays ; TK2 inhibitors
- 刊名：Biochemical and Biophysical Research Communications
- 出版年：2006
- 出版时间：26 May 2006
- 年：2006
- 卷：344
- 期：1
- 页码：30-36
- 全文大小：259 K
文摘
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− 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-(β-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.