Bromovinyl-deoxyuridine: A selective substrate for mitochondrial thymidine kinase in cell extracts.
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-(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.
Franzolin
Elisa
E
Department of Biology, University of Padova, 35131 Padova, Italy.
Rampazzo
Chiara
C
Pérez-Pérez
María-Jesús
MJ
Hernández
Ana-Isabel
AI
Balzarini
Jan
J
Bianchi
Vera
V
D001087:Arabinonucleosides / Q000378:metabolism; D001681:Biological Assay; D001973:Bromodeoxyuridine / Q000031:analogs & derivatives* / Q000378:metabolism; D002457:Cell Extracts / Q000737:chemistry; D005347:Fibroblasts / Q000201:enzymology; D006801:Humans; D008928:Mitochondria / Q000201:enzymology*; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity; D013936:Thymidine / Q000031:analogs & derivatives / Q000378:metabolism; D013937:Thymidine Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D001087:Arabinonucleosides; D002457:Cell Extracts; D047428:Protein Kinase Inhibitors; C020235:brivudine; C105339:thymidine kinase 2; D013937:Thymidine Kinase; C092717:thymidine kinase 1; D001973:Bromodeoxyuridine; C010032:thymine arabinoside; D013936:Thymidine