Large polypeptides of 10S DNA polymerase α from calf thymus: rapid isolation using monoclonal antibody and tryptic peptide mapping analysis

• Published in: Nucleic acids research 1984-06, Vol.12 (11), p.4455-4467
• Main Authors: Masaki, Shigeo, Tanabe, Kazushi, Yoshida, Shonen
• Format: Article
• Language: English
• Subjects: Animals; Antibodies, Monoclonal; Antigen-Antibody Complex - analysis; calf thymus; Cattle; DNA polymerase I; DNA Polymerase II - isolation & purification; Molecular Weight; Peptide Fragments - analysis; peptide mapping; Peptides - isolation & purification; Thymus Gland - enzymology; Trypsin
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/12.11.4455

The polypeptides recognized by a monoclonal antibody against calf thymus DNA polymerase α (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 α 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 α. 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 α 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.