A continuous spectrophotometric assay for thymidine and deoxycytidine kinases.
We have developed a continuous spectrophotometric assay for thymidine and deoxycytidine kinase activities by coupling nucleoside 5'-monophosphate formation to a methylation reaction which generates a product absorbing at 340 nm. With thymidine kinase, we used the alternate substrate deoxyuridine and coupled the reaction to thymidylate synthase. For deoxycytidine kinase, we coupled the reaction to a thymidylate synthase mutant which converts the product 2'-deoxycytidine-5'-monophosphate (dCMP) to m5dCMP. In both cases, the methylation reactions are accompanied by conversion of 5,10-methylene-5,6,7,8-tedrahydrofolate to 7,8-dihydrofolate and can be continuously monitored by the increase of absorbance at 340 nm. The assay should be particularly useful for kinetic studies, and for the purification of these enzymes from various sources.
Liu
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Department of Biochemistry and Biophysics, University of California, San Francisco 94143-0448, USA.
Santi
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DV
D003842:Deoxycytidine Kinase / Q000378:metabolism*; D003843:Deoxycytidine Monophosphate / Q000378:metabolism; D003856:Deoxyuracil Nucleotides / Q000378:metabolism; D003857:Deoxyuridine / Q000378:metabolism; D005492:Folic Acid / Q000031:analogs & derivatives / Q000032:analysis; D018259:Herpesvirus 1, Human / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008745:Methylation; D013053:Spectrophotometry / Q000379:methods*; D013763:Tetrahydrofolates / Q000378:metabolism; D013937:Thymidine Kinase / Q000378:metabolism*; D013940:Thymidylate Synthase / Q000235:genetics / Q000378:metabolism*
D003856:Deoxyuracil Nucleotides; D013763:Tetrahydrofolates; C013123:5,10-methylenetetrahydrofolic acid; D003843:Deoxycytidine Monophosphate; C010920:dihydrofolate; D005492:Folic Acid; C007267:2'-deoxyuridylic acid; C075106:deoxycytidylate methylase; D013940:Thymidylate Synthase; D013937:Thymidine Kinase; D003842:Deoxycytidine Kinase; D003857:Deoxyuridine