Purification and some properties of polynucleotide kinase from rat liver nuclei.

Abstract

Polynucleotide kinase was purified from crude extracts of rat liver nuclei by affinity chromatography on DNA agarose. At optimal pH (5.5) and at saturating concentrations of ATP and DNA, the purified enzyme was found to express maximal activity in the presence of 0.10-0.15 M NaCl; higher salt concentrations inhibited the activity. At the optimal pH and NaCl concentration, the apparent KM for 5'-OH-DNA at 100 microM ATP was 46.2 microM and the apparent KM for ATP at 1 mM 5'-OH-DNA was 15.8 microM. Polynucleotide kinase was protected against heat inactivation by ATP as well as by 5'-OH-DNA at low and moderately high NaCl concentrations, which suggests that under these conditions the enzyme reacts according to a random reaction mechanism. Studies on the heat inactivation of the enzyme in the presence of 5'-OH- or 5'-P-DNA revealed the protection occurs only if 5'-OH-DNA is present, at NaCl concentrations permitting the enzyme to bind DNA.