5-bromovinyl 2'-deoxyuridine phosphorylation by mitochondrial and cytosolic thymidine kinase (TK2 and TK1) and its use in selective measurement of TK2 activity in crude extracts

Mitochondrial thymidine kinase (TK2) is responsible for phosphorylation of thymidine and deoxycytidine and plays a crucial role in mitochondrial DNA precursor synthesis. TK2 is expressed in all tissues at low levels complicating accurate determinations, especially in tissues with high cytosolic thymidine kinase (TK1) activity. Recently, 5-bromovinyl 2'-deoxyuridine (BvdU) at 0.2 mu M was used to measure TK2 activity selectively. BvdU phosphorylation by pure human TK2 and TK1 was tested here, and the ratio of BvdU phosphorylation by TK2/TK1 was 91 at 0.2 mu M but was 500 at 2.5 mu M. Therefore, for reliable measurement of TK2 activity higher BvdU concentration should be used.