Chick embryo DNA polymerase alpha. Polypeptide components and their microheterogeneity.
DNA polymerase alpha was purified from a chick embryo extract by ammonium sulfate fractionation and successive column chromatographies. The final preparation had a specific enzyme activity of 39,000 units/mg of protein with activated calf thymus DNA as a template.primer. Electrophoresis in nondenaturing polyacrylamide gel showed that the preparation contained two proteins, one of which was associated with DNA polymerase activity. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the electrophoretically pure enzyme gave two clusters of polypeptide bands. One was composed of 3-4 polypeptides with similar electrophoretic mobilities corresponding to Mr = 130,000-155,000, and the other consisted of four distinct polypeptide bands corresponding to Mr = 59,000, 56,000, 54,000, and 51,000. Two-dimensional tryptic peptide mapping analysis of these polypeptides after 125I-iodination indicated that the structures of all four Mr = 51,000-59,000 polypeptides were very similar. In addition, polypeptides in the Mr = 145,000-155,000 region had almost identical structure with those in the Mr = 130,000-140,000 region. No significant structural homology was observed between the high molecular weight polypeptides and low molecular weight polypeptides. These findings indicate that the four polypeptides of Mr = 51,000-59,000 are generated by minor modification(s) of a single polypeptide. Similarly, the heterogeneity of the high molecular weight polypeptides is brought about by minor modification(s). Thus, chick embryo DNA polymerase alpha is basically composed of two kinds of subunit polypeptides.
D000818:Animals; D002642:Chick Embryo; D004257:DNA Polymerase II / Q000302:isolation & purification* / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis; D010455:Peptides / Q000032:analysis; D014357:Trypsin
D010446:Peptide Fragments; D010455:Peptides; D004257:DNA Polymerase II; D004259:DNA-Directed DNA Polymerase; D014357:Trypsin