A rapid purification of T4 polynucleotide kinase using Blue Dextran-Sepharose chromatography.
A rapid batch procedure is described for purification of T4 polynucleotide kinase (ATP:5'-dephosphopolynucleotide 5'-phosphotransferase, EC 2.7.1.78) to near homogeneity using Blue Dextran-Sepharose chromatography. The enzyme preparation is sufficiently free of contaminating endonuclease and alkaline phosphatase activities to be suitable for radioactively labeling nucleic acids in vitro. Kinetic measurements indicate that the chromophore of Blue Dextran, Cibacron Blue F3GA, inhibits the activity of T4 polynucleotide kinase competitively with respect to single stranded DNA substrate and non-competitively with respect to the rATP substrate.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000873:Anthracenes; D001665:Binding Sites; D001667:Binding, Competitive; D002847:Chromatography, Agarose; D003090:Coliphages / Q000201:enzymology*; D004396:Coloring Agents; D004277:DNA, Single-Stranded / Q000378:metabolism; D003911:Dextrans / Q000378:metabolism / Q000494:pharmacology; D010770:Phosphotransferases / Q000302:isolation & purification*; D011116:Polynucleotide 5'-Hydroxyl-Kinase / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D014227:Triazines
D000873:Anthracenes; D004396:Coloring Agents; D004277:DNA, Single-Stranded; D003911:Dextrans; D014227:Triazines; C012842:Cibacron Blue F 3GA; D000255:Adenosine Triphosphate; D010770:Phosphotransferases; D011116:Polynucleotide 5'-Hydroxyl-Kinase