Large polypeptides of 10S DNA polymerase alpha from calf thymus: rapid isolation using monoclonal antibody and tryptic peptide mapping analysis.
The polypeptides recognized by a monoclonal antibody against calf thymus DNA polymerase alpha (secreted from a hybridoma CL22 -2- 42B , Nucleic Acids Res. (1982) 10, 4703-4713) were identified by the immunoblot method as the large polypeptides of the partially-purified 10S DNA polymerase alpha fraction. Using an immunoprecipitation technique with the monoclonal antibody, a rapid immunological isolation of the polypeptides has been achieved. By this method, the large polypeptides with Mr = 140,000, 145,000, and 150,000 were isolated from a partially-purified preparation of 10S DNA polymerase alpha. On the other hand, the polypeptides with Mr = 150,000, 180,000, and 240,000 were obtained from a crude extract of calf thymus. Tryptic peptide maps showed that the large polypeptides with Mr = 150,000, and 180,000 were very similar in primary structure and that the structures of Mr = 180,000 and 240,000 polypeptides contained partially common sequences. Among these polypeptides, the Mr = 150,000 polypeptide was shown to correlate with the enzyme activity. These results suggest that the large polypeptide of 10S DNA polymerase alpha is initially synthesized as Mr = 180,000 or larger polypeptide, then converted to the form with Mr = 150,000. The Mr = 140,000 and 145,000 polypeptides in the purified preparation may be artificial products formed during purification.
D000818:Animals; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex / Q000032:analysis; D002417:Cattle; D004257:DNA Polymerase II / Q000302:isolation & purification*; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis; D010455:Peptides / Q000302:isolation & purification; D013950:Thymus Gland / Q000201:enzymology*; D014357:Trypsin
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D010446:Peptide Fragments; D010455:Peptides; D004257:DNA Polymerase II; D014357:Trypsin