Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.1.21 (thymidine kinase)
 7,561 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Alkylated nucleotides have been detected by 32P-postlabelling using the enzyme T4 polynucleotide kinase which phosphorylates the 3'-mononucleotides to give the 3',[5'-32P]bisphosphates. These may then be separated by two-dimensional TLC as the bisphosphates or the [5'-32P]monophosphates. We describe here an alternative approach using the Epstein-Barr virus (EBV) encoded thymidine kinase (TK) to directly phosphorylate adducted nucleosides to give the [5'-32P]monophosphates. Using a series of methyl, ethyl and butyl thymidines EBV-encoded TK was shown to phosphorylate a wide range of adducted thymidines with varying degrees of labelling efficiency; N3-methyl thymidine was labelled with the highest efficiency and O4-ethyl thymidine the lowest. Whereas O4-methyl thymidine was labelled at a higher efficiency than O2-methyl thymidine, O4-ethyl and O4-butyl thymidines were labelled at a much lower efficiency than the corresponding O2-alkyl thymidines. Labelling efficiency increased with pH in the range pH 7 to pH 9, but the relative labelling efficiency was ATP independent. This direct phosphorylation of adducted nucleosides offers an alternative approach to the detection of alkylated residues in DNA which may complement current postlabelling procedures.
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PMID:32P-postlabelling of alkylated thymidines using Epstein-Barr virus encoded thymidine kinase. 184 71

3'-Azido-3'-deoxythymidine (AZT) was the first anti-retroviral nucleoside analog to be used in the treatment and prevention of acquired immunodeficiency syndrome (AIDS). A novel (32)P-postlabeling assay, based upon thymidine kinase (TK) instead of the conventional T(4) polynucleotide kinase, has been developed for the detection of the levels of AZT incorporated into DNA. After enzymatic digestion of DNA to deoxynucleoside 3'-monophosphates, AZT was isolated by ethyl acetate extraction. The ethyl acetate was evaporated and the AZT was postlabeled by 5'-phosphorylation with [gamma-(32)P]ATP and TK. AZT was detected at a level of 51.5+/-6.3 (mean+/-SD; n=4) AZT molecules/10(5) nucleotides following in vitro incorporation of the drug into high-molecular-weight rat-liver DNA. The (32)P-postlabeling method was further validated by the detection of AZT in lung and liver DNA from neonatal mice treated with AZT. The levels of AZT in lung and liver DNA were proportional to the dose, with the levels in lung DNA being two-fold higher than those for liver DNA. The limit of detection for the assay was 8 AZT molecules/10(7) nucleotides using 10 microg of DNA.
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PMID:Development of a novel (32)P-postlabeling method for the analysis of 3'-azido-3'-deoxythymidine. 1077 26