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.
Koivisto
P
P
Institute of Occupational Health, Helsinki, Finland.
Hemminki
K
K
D000478:Alkylation; D003848:Deoxyguanine Nucleotides / Q000378:metabolism*; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011116:Polynucleotide 5'-Hydroxyl-Kinase / Q000032:analysis
D003848:Deoxyguanine Nucleotides; D010761:Phosphorus Radioisotopes; C007257:2'-deoxyguanosine 5'-phosphate; D011116:Polynucleotide 5'-Hydroxyl-Kinase