Immunochemical analysis of molecular forms of mammalian DNA ligases I and II.
Using specific antibodies against calf thymus DNA ligases I and II (EC 6.5.1.1), we have investigated the polypeptide structures of DNA ligases I and II present in the impure enzyme preparations, and estimated the polypeptides of DNA ligases I and II present in vivo. Immunoblot analysis of DNA ligase I after sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a 130-kDa polypeptide as a major one in the enzyme preparations from calf thymus throughout the purification. In addition to the 130-kDa polypeptide, a 200-kDa polypeptide was detected in the enzyme preparations at the earlier steps of the purification, and a 90-kDa polypeptide was observed as a minor one in the enzyme preparations at the later steps of the purification. The polypeptides with molecular weight of 130 000 and 90 000 were detected by SDS-polyacrylamide gel electrophoresis of DNA ligase I-[3H]AMP complex. These results suggest that a 200-kDa polypeptide of DNA ligase I present in vivo is degraded to a 130-kDa polypeptide and then to a 90-kDa polypeptide during the isolation and purification procedures. On the other hand, the monospecific antibody against calf thymus DNA ligase II cross-reacted with only a 68 kDa polypeptide in the enzyme preparations throughout the purification, suggesting that the 68-kDa polypeptide is a single form of calf thymus DNA ligase II present in vivo as well as in vitro.
D000818:Animals; D002286:Carcinoma, Ehrlich Tumor / Q000201:enzymology; D002417:Cattle; D003101:Collodion; D011088:DNA Ligases / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D006651:Histocytochemistry; D007158:Immunologic Techniques; D008970:Molecular Weight; D011118:Polynucleotide Ligases / Q000302:isolation & purification*; D013950:Thymus Gland / Q000201:enzymology
D003101:Collodion; D011088:DNA Ligases; D011118:Polynucleotide Ligases