[Covalent labelling of the Klenow fragment of DNA-polymerase I from E. coli].
Incubation of the Klenow fragment of E. coli DNA polymerase I with [alpha-32P] dNTP (or NTP) results in the covalent radiolabelling of the enzyme, the bond being stable in acid (pH 2) and alkaline (pH 12) conditions and nucleophiles, such as beta-mercaptoethylamine, efficiently inhibiting the labelling. It is suggested that radiolabelling of the enzyme is the result of formation of chemically active products of the radiolysis of [alpha-32P]NTP (which are likely to be radicals). Non-radioactive NTP hinder the labelling, whereas Mg2+ and polynucleotide do not affect it. Cleavage of the enzyme by hydroxylamine and cyanogen bromide and analysis of gel-electrophoretic patterns of the cleavage products led to conclusion that 32P-label is located between Gly-544 and Met-647.
Degtiarev
S Kh
SKh
Zaĭchikov
E F
EF
Lavruk
O I
OI
Mitina
R L
RL
Mustaev
A A
AA
Rikhter
V A
VA
D002851:Chromatography, High Pressure Liquid; D004256:DNA Polymerase I / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D009711:Nucleotides / Q000378:metabolism; D013379:Substrate Specificity
D009711:Nucleotides; D004256:DNA Polymerase I; D004259:DNA-Directed DNA Polymerase
Kovalentnoe mechenie fragmenta Klenova DNK-polimerazy I E. coli.