The purification of T4 polynucleotide kinase results in the copurification of an activity which′will specifically remove the 3′-terminal phosphate from a variety of deoxyribonucleotides and ribonucleotides in the absence of ATP. This phosphatase activity requires magnesium, has a pH optimum of 6.0, and is more active with deoxyribonucleotides than ribonucleotides. T4 polynucleotide kinase and the 3′-phosphatase activity copurify by gradient elution column chromatography on DEAE-cellulose, phosphocellulose, and hydroxylapatite. The two activities are included in and comigrate on Sephadex G-200. Polyacrylamide gel electrophoresis at pH 9.2 results in comigration of the two activities together with the major protein band. The two activities respond in parallel to heat inactivation at 35 °C and ATP, a substrate for the kinase only, protects both activities from heat inactivation. It is therefore suggested that the two activities are functions of the same protein molecule.
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