AraUTP-Affi-Gel 10: a novel affinity absorbent for the specific purification of DNA polymerase alpha-primase.
For the specific purification of eukaryotic DNA-dependent DNA polymerase alpha, we prepared two novel affinity resins bearing 5-(E)-(4-aminostyryl) araUTP as a ligand. One of them was araUTP-Sepharose 4B which was coupled directly with the ligand and the other was araUTP-Affi-Gel 10 which was coupled with the ligand through a spacer. No DNA polymerase alpha-primase activity from cherry salmon (Oncorhynchus masou) testes was bound on the araUTP-Sepharose 4B in all cases examined. On the other hand, the araUTP-Affi-Gel 10 retains this enzyme activity when poly(dA) or poly(dA)-oligo(dT)12-18 is present. The retained enzyme activity was sharply eluted around 100-mM KCl concentrations as a single peak, and this fraction showed a specific activity of about 170,000 units/mg as alpha-polymerase activity. The highly purified DNA polymerase alpha-primase isolated using the araUTP-Affi-Gel 10 contained only three polypeptides, which showed Mr values of 120,000, 62,000, and 58,000, respectively, as judged using sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Izuta
S
S
Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan.
Saneyoshi
M
M
D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods*; D004257:DNA Polymerase II / Q000302:isolation & purification; D019915:DNA Primase; D008297:Male; D008970:Molecular Weight; D010455:Peptides / Q000302:isolation & purification; D012316:RNA Nucleotidyltransferases / Q000302:isolation & purification*; D012474:Salmon; D012685:Sepharose / Q000031:analogs & derivatives*; D013737:Testis / Q000201:enzymology; D014500:Uracil Nucleotides / Q000031:analogs & derivatives; D014544:Uridine Triphosphate / Q000031:analogs & derivatives
D010455:Peptides; D014500:Uracil Nucleotides; C058464:ara-UTP-Affi-Gel 10; D012685:Sepharose; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases; D004257:DNA Polymerase II; D014544:Uridine Triphosphate