32P-postlabelling of 2-hydroxyethylated, ethylated and methylated adducts of 2'-deoxyguanosine 3'-monophosphate.
Ethylene oxide, diethyl sulphate and dimethyl sulphate were used to synthesize the corresponding 7-alkylation products of 2'-deoxyguanosine 3'-monophosphate (dGMP). The purified adducts were used as substrates in the 32P-post-labelling reaction with T4 polynucleotide kinase. The kinetics of phosphorylation were studied with 7-(2-hydroxyethyl)-dGMP: most net product was formed by 15 min and only a small increase was seen until 4 h. When different concentrations of the adducts were tested, a complete phosphorylation was noted for 7-methyl-dGMP to the lowest tested amount of 1 fmol. The efficiencies of phosphorylation for 7-ethyl- and 7-hydroxyethyl-dGMP were 1.5 and 0.5% respectively. The proportions phosphorylated were uniform over the concentration range tested. The results demonstrate dramatic differences in the efficiency of phosphorylation between structural analogues, which is probably related to a decreased affinity of the substrate to the enzyme or to an interference in the transfer of the phosphate group on the active site of the enzyme.[1]References
- 32P-postlabelling of 2-hydroxyethylated, ethylated and methylated adducts of 2'-deoxyguanosine 3'-monophosphate. Koivisto, P., Hemminki, K. Carcinogenesis (1990) [Pubmed]
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