DNA-dependent adenosinetriphosphatase A: immunoaffinity purification and characterization of immunological reagents.
We describe an immunoaffinity purification of DNA-dependent ATPase A from fetal calf thymus. The rapid purification increases the yield of enzymatically active enzyme approximately 4-fold, with up to a 7-fold increase in specific activity, and significantly improves the yield of a higher molecular weight species of ATPase A. In the presence of a denatured calf thymus DNA effector, the immunoaffinity-purified enzyme has a specific activity that is more than 10-fold higher than reported for any other eukaryotic DNA-dependent ATPase and 100-fold higher than most others. The improvement in yield has allowed several polypeptides to be identified using monoclonal antibodies, and these polypeptides are demonstrated to be structurally related by partial peptide mapping with N-chlorosuccinimide. The preferred DNA effector for ATP hydrolysis continues to be a DNA primer-template junction with an adjacent stretch of single-stranded DNA. We have used the immunoaffinity-purified enzyme to develop additional stable murine hybridoma monoclones, resulting in a bank of antibodies that recognize a number of different epitopes. All of the monoclonal antibodies react with both calf thymus DNA-dependent ATPase A and bacteriophage T4 gene 44 protein, a DNA-dependent ATPase essential for DNA replication in the bacteriophage T4 system. These monoclonal antibodies should facilitate the development of our understanding with respect to the role and regulation of DNA-dependent ATPases in eukaryotic DNA replication.
Department of Biochemistry, University of Virginia Health Sciences Center, Charlottesville 22908.
L D
LD
Mesner
P A
PA
Truman
J W
JW
Hockensmith
D000251:Adenosine Triphosphatases / Q000276:immunology / Q000302:isolation & purification*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D002417:Cattle; D002846:Chromatography, Affinity / Q000379:methods; D002848:Chromatography, DEAE-Cellulose; D003429:Cross Reactions; D004265:DNA Helicases / Q000276:immunology / Q000302:isolation & purification*; D004259:DNA-Directed DNA Polymerase / Q000276:immunology; D004591:Electrophoresis, Polyacrylamide Gel; D007202:Indicators and Reagents; D051379:Mice; D013950:Thymus Gland / Q000201:enzymology; D014764:Viral Proteins / Q000276:immunology
D000911:Antibodies, Monoclonal; D007202:Indicators and Reagents; D014764:Viral Proteins; C074855:gene 44 protein, Enterobacteria phage T4; D004259:DNA-Directed DNA Polymerase; D000251:Adenosine Triphosphatases; D004265:DNA Helicases