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

Abstract

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 μ 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 μ M but was 500 at 2.5 μ M. Therefore, for reliable measurement of TK2 activity higher BvdU concentration should be used.

Keywords:

• Thymidine kinase 2
• thymidine kinase 1
• activity determination
• bromovinyl deoxyurindine

This work was supported by a grant from the Swedish Research Council.

Notes

∗Pure recombinant human TK1 and TK2 were used in the assays using [³H]-BvdU as substrate at concentration range of 1 to 160 μ M. With TK2 the substrate saturation curve followed Michaelis-Menten equation while with TK1 the activity was linear in the concentration range and did not reach saturation. The ratio was calculated from the activity at each concentration for TK1 and TK2.

∗Assays were performed with mouse liver and spleen extracts using either 3H-BvdU or 3H-dThd as substrate at various concentration. The experiments were done in duplicates of extracts from three individual mice. The results are mean ± SE.

∗Total TK activity was determined by using 100 μ M [³H]-dThd. TK2 activity was estimated from assays with [³H]-dThd in the presence of excess dCyd (1 mM). If the inhibition by dCyd is > 50% inhibition then the total TK activity equals TK2 activity. If the inhibition by dCyd is < 40% the level of TK1 in the sample is too high and TK2 activity cannot be estimated, for example, in the spleen. In such case TK2 activity can be determined by using [³H]-BvdU (2.5 μ M) in the presence of 1 mM 6-amino 5-chlorouracil.

^# TK2 activity could not be estimated due to the presence of high TK1 activity.