Purification and characterization of DNA ligase II from Drosophila melanogaster.
Drosophila melanogaster contains DNA ligases I and II. The activity of DNA ligase I is especially high during early embryonic periods, but decreases rapidly afterwards. Although the activity of DNA ligase II is low, it persists throughout all developmental stages. The specific activity of DNA ligase II is high in embryos, but the total activity per body mass was highest in pupae. To characterize the properties of DNA ligase II further and to clarify its differences from DNA ligase I, DNA ligase II was prepared from pupae of D. melanogaster. The enzyme was purified about 3200-fold by ammonium sulfate fractionation (40-70% saturation), phosphocellulose (P11) and Ultrogel column chromatography. Some of the properties have been reported previously. The isoelectric point of DNA ligase II was 6.4 while those of DNA ligase I were 4.9 and 5.8. The optimum pH of DNA ligase II was 7.8-8.1 but 8.0-8.5 for DNA ligase I. The molecular masses of DNA ligase II adducts with AMP were determined as 90 and 70 kDa. These adducts were degraded to 42 and 14.4 kDa by trypsin digestion. For preparation of monoclonal antibodies, a mouse was immunized with the purified enzyme. Two clones, 10-6 and 3-3 IgM, were obtained and purified from mouse ascites. These antibodies showed both binding and neutralizing activities toward DNA ligase II from D. melanogaster, but did not react with DNA ligase I from the same origin. These results showed clearly that DNA ligases I and II have different properties and suggest they have different roles during the developmental stages of D. melanogaster.
Takahashi
M
M
Mitsubishi Kasei Institute of Life Sciences, Minamiooya, Japan.
Tomizawa
K
K
D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis; D000918:Antibody Specificity; D003429:Cross Reactions; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000201:enzymology*; D007526:Isoelectric Point; D007814:Larva / Q000201:enzymology; D010446:Peptide Fragments / Q000032:analysis; D014357:Trypsin
D000911:Antibodies, Monoclonal; D010446:Peptide Fragments; D014357:Trypsin; D011088:DNA Ligases; D000072481:DNA Ligase ATP