Quantitative and kinetic examination of 32P-postlabeling of etheno-substituted nucleotides.
1,N6-ethenodeoxyadenosine-, 1,N2-ethenodeoxyguanosine- and 3,N4-ethenodeoxycytidine-3'-monophosphates were labeled by [gamma-32P] ATP using T4 polynucleotide kinase in conditions commonly used for the 32P-postlabeling assay. Kinetic studies showed that the reaction is fast reaching a plateau after 15-30 min. The efficiency of phosphorylation, as studied by substrate-product concentration dependency, was between 50-100% at the lower substrate concentrations. The adducts are labeled efficiently at sub-femtomole levels. All the adducts were sensitive to the 3'-dephosphorylation by P1 nuclease although the guanine derivative appeared to be more resistant than the two other adducts.
Institute of Occupational Health, Helsinki, Finland.
K
K
Szyfter
K
K
Hemminki
A E
AE
Crane
W P
WP
Watson
D000255:Adenosine Triphosphate; D003843:Deoxycytidine Monophosphate / Q000031:analogs & derivatives* / Q000138:chemical synthesis; D003848:Deoxyguanine Nucleotides / Q000138:chemical synthesis*; D007553:Isotope Labeling / Q000379:methods; D007700:Kinetics; D015394:Molecular Structure; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011116:Polynucleotide 5'-Hydroxyl-Kinase / Q000378:metabolism; D013604:T-Phages / Q000201:enzymology
D003848:Deoxyguanine Nucleotides; D010761:Phosphorus Radioisotopes; D003843:Deoxycytidine Monophosphate; C071135:3,N(4)-etheno-3'-deoxycytidine monophosphate; C071136:3,N(4)-etheno-3'-deoxyguanosine monophosphate; D000255:Adenosine Triphosphate; D011116:Polynucleotide 5'-Hydroxyl-Kinase