Purification of DNA ligase II from calf thymus and preparation of rabbit antibody against calf thymus DNA ligase II.
DNA ligase II has been purified about 4,000-fold to apparent homogeneity from a calf thymus extract. The ligase consists of a single polypeptide with a molecular weight of 68,000 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. On fluorography after electrophoresis, a DNA ligase-[3H]AMP complex gave a single band corresponding to a molecular weight of 68,000. The Km values of the ligase for ATP and nicked DNA (5'-phosphoryl ends) were obtained to be 40 and 0.04 microM, respectively. Antibody against calf thymus DNA ligase II was prepared by injecting the purified enzyme into a rabbit. The antibody cross-reacted with DNA ligase II but not with calf thymus DNA ligase I. DNA ligase II was not affected by antibody against calf thymus DNA ligase I with a molecular weight of 130,000 (Teraoka, H. and Tsukada, K. (1982) J. Biol. Chem. 257, 4758-4763). These results indicate that DNA ligase II (Mr = 68,000) is immunologically distinct from DNA ligase I (Mr = 130,000).
D000818:Animals; D000906:Antibodies; D000936:Antigen-Antibody Complex; D002417:Cattle; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000302:isolation & purification* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005779:Immunodiffusion; D007700:Kinetics; D008970:Molecular Weight; D011118:Polynucleotide Ligases / Q000302:isolation & purification*; D013950:Thymus Gland / Q000201:enzymology*
D000906:Antibodies; D000936:Antigen-Antibody Complex; D011088:DNA Ligases; D011118:Polynucleotide Ligases; D000072481:DNA Ligase ATP