Purification and characterization of DNA polymerase alpha from plasmodia of Physarum polycephalum.
DNA polymerase alpha from Physarum polycephalum has been purified from freshly harvested microplasmodia. An inhibitory activity was removed by precipitation with poly(ethyleneimine) and interfering type-beta-like DNA polymerase by chromatography on phosphocellulose. The preparation was free of endonucleases and exonucleases. The DNA-polymerizing polypeptide had a molecular mass of 140 kDa 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 in high-molecular-mass forms. The type of isolated DNA polymerase was established on the basis of inhibition and template-primer utilization experiments underlying the classification of DNA polymerases from higher eucaryotes. The majority of the DNA-polymerizing activity was contained in the cell nucleus fraction and was inhibited by aphidicolin. The isoelectric point (pI) was 6.7 +/- 0.2, the pH optimum at pH 6.8, and the temperature optimum at 40 degrees C. Monovalent salts, Li+, Na+, NH+4, K+, were inhibitory except for small activation maxima at 10 mM, 75 mM and 100 mM in the case of Na+, NH+4 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 replicons in plasmodia and with the number of molecules of DNA polymerases in higher eucaryotes.
Weber
C
C
Institut für Biophysik und Physikalische Biochemie der Universität, Regensburg, Federal Republic of Germany.
Fischer
H
H
Holler
E
E
D002467:Cell Nucleus / Q000201:enzymology; D003593:Cytoplasm / Q000201:enzymology; D004257:DNA Polymerase II / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D008970:Molecular Weight; D010804:Physarum / Q000201:enzymology*; D011489:Protein Denaturation; D013698:Templates, Genetic
D004257:DNA Polymerase II