Purification and caracterization of DNA polymerase α from plasmodia of Physarum polycephalum

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Abstract
DNA polymerase α from Physarum polycephalum has been purified fromfreshly harvested microplasmodia. An inhibitory activity was removed by precippitation with poly(ethyleneimine) and interfering type‐β‐like DNA polymerase by chromatography on phosphocellulose. The preparation was free of endonucleases and exonucleases.The DNA‐polymerizing polypeptide had a molecular mass of 140kDa by polyacrylamide gel electrophoresis under denaturing conditions. It was contained in purified samples and in crude cell extracts. Peptides of smaller size that reacted with antibodies against this protein were generated during purification and prolonged standing. Molecular sizing under non‐denaturing conditions resulted inhigh‐molecular‐mass forms.The isoelectric point (pI) was 6.7 ± 0.2, the pH optimum at pH 6.8, and the temperature optimum at 40°C. Monovalent salts, Li+, Na+, NH4+, K+, were inhibitory except for small activation maxima at 10mM, 75 mM and 100 mM in the case of Na+, NH4+ and K+ respectively. The bivalent cations Mg2+ and Mn2+ had broad activity maxima at 3‐20 mM concentrations, which were shifted to 0.05‐0.1 mM in the case of Mn2+ and synthetic DNA homopolymers.The numbers of molecules of DNA polymerases in Physarum nuclei were calculated and compared with the established number of molecules of DNA polymerasesinhigher eucaryotes